XB-ART-36588Differentiation 2008 Apr 01;764:392-403. doi: 10.1111/j.1432-0436.2007.00229.x.
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The efficiency of Xenopus primordial germ cell migration depends on the germplasm mRNA encoding the PDZ domain protein Grip2.
A microarray analysis of vegetal pole sequences in the egg and early Xenopus laevis embryo identified Unigene Xl.14891 as a vegetally localized RNA. Analysis of the Xenopus tropicalis genome showed this Unigene to be localized near the 3' end of the Grip2 (glutamate receptor interacting protein 2) transcription unit. RACE showed that the Unigene represented the 3' UTR of Grip2 mRNA. Grip2 mRNA is present in the mitochondrial cloud of late pre-vitellogenic oocytes and then in the germplasm through oogenesis and early development until tailbud tadpole stages. Interference with Grip2 mRNA translation using two antisense morpholino oligos (MOs) impairs primordial germ cell (PGC) migration to the germinal ridges. Both MOs also inhibit swimming movements of the tailbud tadpole, known to involve glutamate receptors. We conclude that Grip2 has several functions in the embryo, including enabling efficient PGC migration.
PubMed ID: 17924960
Article link: Differentiation
Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: cyp26a1 grip1 grip2 myc pgat pgc
Antibodies: Myc Ab3 Tubg1 Ab2
Morpholinos: grip2 MO3 grip2 MO4
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|Fig. 2 Distribution of Grip2 mRNA shown by in situ hybridization. (A) Two stage I oocytes. (B) Stage II oocyte. (C) Late stage III oocyte. (D) Stage VI oocyte, vegetal view. (E) Stage 9 blastula. (F) Stage 26. (G) Stage 28, (G0) with the migrating primordial germ cells (PGCs) enlarged (arrows). (H) Stage 37, when the PGCs were no longer revealed by the Grip2 probe, whereas they were by Xpat (I, I0). The embryos, but not the oocytes, were bleached. The scale bars represent 100 um.|
|grip2 (glutamate receptor interacting protein 2 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage oocyte III.|
|grip2 (glutamate receptor interacting protein 2 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 9, mid sagittal section.|
|grip2 (glutamate receptor interacting protein 2 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage oocyte VI, vegetal hemisphere view.|
|Fig. 3 Inhibition of Grip2 mRNA translation by morpholino oligo MO1. Where indicated, embryos were injected into the vegetal pole with 30 ng Grip1 MO1 at the early 2-cell stage, followed by 200 pg of Grip2 mRNA with its own 50 UTR and a C-terminal Myc tag. Five embryos were homogenized at stage 9 or 37 and subjected to SDS gel electrophoresis and Western blotting with an anti-Myc antibody.|
|Fig. 4 Effects of an antiGrip2 morpholino oligo (MO) injected into the vegetal pole at the two-cell stage on embryo morphology and primordial germ cells (PGCs). (A). Stage 33 uninjected embryos, (B, C) compared with those with 30 ng MO1. (D) A stage 39 embryo injected with 30 ng MO1, showing an abnormal fin. (E–H) Stage 41 embryos; (E) uninjected, (F) injected with 10 ng, (G, H) injected with 30 ng MO1. Note the lateral curves in (H) and also (B). (I–N) In situ hybridisations to Xpat mRNA in PGCs at stages 37 (I– K) and 39/40 (L–N). These embryos were control (I,L) or injected with 10 ng (J,) or 30 ng (K,N) MO1, or 40 ng control MO (M). (L) The arrow shows how even in control, uninjected embryos there may be very few PGCs delayed in migration.|
|Fig. 5 Effects on morphology and primordial germ cells of a second morpholino oligo, (grip MO4) MO2. (A–E) Effects on overall morphology at stage 33. (A) Uninjected. (B) 50 ng control MO. (C) 30 ng MO2. (D) 50 ng MO2. Note the curled embryos, as if still unhatched. (E) 100 ng MO2. (F–L) Xpat in situ hybridizations to stage 39 embryos injected with MO2. (F) Uninjected. (G) 50 ng control MO. (H) 30 ng MO2. (I–K) 50 ng MO2. (L) 100 ng MO2.|
|Fig. 6 Effect of Grip2 morpholino oligos (MOs) on the number of primordial germ cells (PGCs) that successfully migrate to the germ ridges. (A) The experiments involve micro-injecting 5 nl FLDx, with or without MOs. (B) Fluorescence image of a stage 45 tadpole injected with FLDx alone. The principal labeling is in the gut (arrow), but the PGCs may also be seen (arrowheads). (C) Fluorescent PGCs in the germ ridges, after dissection. In (B,C), there is a low level of white light to make non-fluorescent structures visible. (D–F) Effect of injected MOs on the number of successfully migrating PGCs. MOs and plasmid DNA was injected into a single vegetal blastomere at the 32-cell stage. The detailed data can be found in Table 1.|
|grip2 (glutamate receptor interacting protein 2 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28,lateral view, anterior left, dorsal up.|
|Fig. 1 (A) Structure of the Grip2 mRNA and protein, showing the PDZ domains. (B) Expression of Grip2 mRNA at different stages according to Affymetrix microarray data (Sinner et al., 2006). (C) Abundance of Grip2 mRNA assayed by RT-PCR.|