Berekelya LA et al. (2007), The protein encoded by the germ plasm RNA Germe...

XB-ART-35368

Differentiation 2007 Jul 01;756:546-58. doi: 10.1111/j.1432-0436.2006.00160.x.

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The protein encoded by the germ plasm RNA Germes associates with dynein light chains and functions in Xenopus germline development.

Berekelya LA , Mikryukov AA , Luchinskaya NN , Ponomarev MB , Woodland HR , Belyavsky AV .

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Germ plasm plays a prominent role in germline formation in a large number of animal taxons. We previously identified a novel maternal RNA named Germes associated with Xenopus germ plasm. In the present work, we addressed possible involvement of Germes protein in germ plasm function. Expression in oocytes followed by confocal microscopy revealed that the EGFP fused to Germes, in contrast to the free EGFP, co-localized with the germ plasm. Overexpression of intact Germes and Germes lacking both leucine zipper motifs (GermesDeltaLZs) resulted in a statistically significant reduction of the number of primordial germ cells (PGCs). Furthermore, the GermesDeltaLZs mutant inhibited PGC migration and produced abnormalities in germ plasm intra-cellular distribution at tailbud stages. To begin unraveling biochemical interactions of Germes during embryogenesis, we searched for Germes partners using yeast two-hybrid (YTH) system. Two closely related sequences were identified, encoding Xenopus dynein light chains dlc8a and dlc8b. Tagged versions of Germes and dlc8s co-localize in VERO cells upon transient expression and can be co-immunoprecipitated after injection of the corresponding RNAs in Xenopus embryos, indicating that their interactions occur in vivo. We conclude that Germes is involved in organization and functioning of germ plasm in Xenopus, probably through interaction with motor complexes.

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Species referenced: Xenopus
Genes referenced: adm dynll1 dynll2 germes h3-3a myc pgat pgc
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	Fig. 1 EGFP-Germes localizes to the germ plasm islands in Xenopus oocytes. Full-grown oocytes were injected with RNA encoding either EGFP alone (A0) or EGFP-Germes (B0). Germ plasm was visualized by staining mitochondria with tetramethylrhodamine ethyl ester (TMRE) (A and B). Merged images (A00 and B00) revealed EGFP-Germes co-localization to germ plasm islands at vegetal cortex of oocytes, while distribution of EGFP alone, although overlapping partially with the mitochondria, is much broader than that of TMRE-stained islands. EGFP expression is not entirely uniform because it is excluded from regions rich in dense yolk platelets. Scale bar510 mm.
	Fig. 2 Overexpression of wild-type (WT) and mutant Germes affects primordial germ cell (PGC) number and quantity of germ plasm. Embryos from all experimental groups were grown until stages 31–33, and following fixation were processed for PGC visualization by in situ hybridization with Xpat probe. PGC numbers were counted for each group, and the embryos were divided into groups according to PGC content (A). The peak of the GermesDLZs- expressing embryos distribution is clearly shifted to groups with lower PGC numbers. Alternatively, embryos were lysed to isolate RNA, and Xpat expression was estimated by Nothern blot hybridization (B). The central panel (C) presents levels of Xpat expression normalized to those of the ubiquitous message EF1a. The identity of lanes 1–5 is shown in (C). Levels of WT and mutant Germes proteins expression were compared with each other as C-terminal fusions to myc-tag (D). The proteins are expressed at similar levels during gastrula (11), neurula (18), and tailbud (27) stages and are not detected later on (stage 33). st., stage.
	Fig. 3 The effects on primordial germ cell (PGC) development of expression of mutant Germes lacking leucine zippers. Germ plasm and PGCs (arrows) in intact (A) and GermesDLZs-expressing embryos (B) have been visualized by whole-mount in situ hybridization with an Xpat probe. Cross-sections through control (C) and mutant (D) embryos demonstrate a PGC on the mesoderm–endoderm border of the control embryo and a PGC residing deep in the endoderm of the mutant embryo. In the PGCs of GermesDLZs-injected embryos, the germ plasm appeared in a pool of yolk-free cytoplasm juxtaposed to the nucleus (F), which is more characteristic for earlier stages, while in PGCs of intact embryos, germ plasm is dispersed between yolk platelets around the nucleus (E). We also always noted the difference in germ plasm color in control and mutant embryos. This may arise from the different amount of Xpat mRNA in the PGCs, which is shown by quantitative Northern blot analysis (Fig. 2). am, axial mesoderm; end, endoderm; lm, lateral mesoderm; n, nucleus; not, notochord; nt, neural tube; pgc, primordial germ cell.
	Fig. 4 Overexpression of wild-type (WT) and mutant Germes affects germ plasm morphology. Embryos from all experimental groups (A, intact; B, water-injected; C, GermesDEFh mRNA-injected; D, GermesDLZs mRNA-injected; E, WT Germes mRNAinjected) were immunostained for Xpat protein (A0–E0). Nuclei were revealed by DAPI-staining; this, to a lower extent, also stains yolk platelets (A–E). Merged images (A00–E00) demonstrate exclusively nuclear Xpat localization in control (A00B00) and GermesDEFh- expressing (C00) embryos. In embryos injected with GermesDLZs (D00) or WT Germes (E00) mRNAs, Xpat is also present in yolk-free cytoplasm around the primordial germ cell (PGC) nucleus. Arrows point to nuclei. Scale bar516 mm.
	Fig. 5 Interaction of Germes with dynein light chains. Sequencing of positive clones found to interact with Germes in yeast two-hybrid (YTH) system revealed them to be homologs of dynein light chains dlc8a and dlc8b. (A) Alignment of murine dlc8a and dlc8b sequences with those of Germes YTH partners in Xenopus. (B–B00) In cultured VERO cells, transiently expressed GFP-Germes (B) and HA-dlc8 (B0) are co-localized, as revealed by the merged image (B00). (C) Germes can be co-immunoprecipitated with dlc8s, but not with histone H3.3. Embryos were injected with mRNAs encoding myc-tagged Germes (MT-G) and HA-dlc8s or HA-H3.3, and lysed at stage 9; immunoprecipitation was performed with anti-myc antibody, Western blot detection with anti-HA antibody. HA-dlc8s (12 kDa) and HA-H3.3 (17 kDa) are expressed in the lysates (1, 8, 9). Both dlc8 proteins are co-precipitated with Germes (2, 4), while they are not detected in the precipitations from lysates expressing dlc8s only (3, 5). 1, 7–9, embryo lysates; 2–6, immunoprecipitations; 1, 2, HA-dlc8b1MT-Germes; 4, 9, HA-dlc8a1MT-Germes; 3, HA-dlc8b; 5, HA-dlc8a; 6, 8, H3.31MT-Germes; 7, uninjected control. Upper bands in precipitations represent immunoglobulins.