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Fig. 1. Analysis of XGRIP2.1 expression by whole mount in situ hybridization and RT-PCR. (A–F) The localization of XGRIP2.1 mRNA in Xenopus oocytes and embryos was analyzed by whole mount in situ hybridization, followed by vibratome sectioning (A′–D′). In stage I oocytes, XGRIP2.1 mRNA was detected in the mitochondrial cloud (A, A′) and at early stage III at the tip of the vegetal cortex (B, B′). At the 2-cell stage of embryogenesis, XGRIP2.1 mRNA is enriched in granular patches of germ plasm at the vegetal pole (C, C′, vegetal pole view). At gastrula stage, the transcript was detected in isolated cells within the endoderm (D, blastoporus view, arrow in panel D′). At tailbud stage, XGRIP2.1 is specific to the migrating germ cells (E, lateral view of the embryo with the head to the left). XGRIP2.1 and Xpat RNAs colocalize in migrating PGCs at tailbud stage of embryogenesis. Embryos were sequentially stained for XGRIP2.1 (blue) and Xpat (red, magnified view in the inset, the first staining of the same embryo) (F, lateral view of the embryo with the head to the left). RT-PCR analysis of the temporal expression during embryogenesis (G) and tissue-specific expression of XGRIP2.1 (H). Total RNA for RT-PCR was prepared from embryos or from adult tissues. Abbreviations: Gv—germinal vesicle, Mc—mitochondrial cloud, Vc—vegetal cortex, Bc—blastocoel, B—brain, O—ovary, E—eye, Sk—skin, M—muscle, F—fat, K—kidney, G—gall bladder, Sp—spleen, I—intestine, T—testes, Li—liver, Sm—stomach, Lu—lung, P—pancreas, H—heart, Sc—spinal cord.
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grip2 (glutamate receptor interacting protein 2)
expression in Xenopus laevis embryo, NF stage 33, assayed via in situ hybridization, lateral view, anterior left, dorsal up.
Primordial germ cells in the embryo stain in blue, confirmed by counterstain for the gene pgat (Primordial germ cell-associated transcript protein) (inset, in red).
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Supplementary Fig. 2.
Fig. 2. XGRIP2.1 MO1 and MO2 inhibit translation of XGRIP2.1 in vitro and in vivo. (A) Reduction or absence of XGRIP2.1 translation was observed in the coupled in vitro transcription/ translation reaction (Promega) upon addition of 1 nmol or 0.1 nmol of either MO1 or MO2, respectively. (B-D) Two-cell stage embryos were injected vegetally into both blastomeres with either 5’UTRXGRIP2.1mycGFP or 5’UTRXGRIP2.1mycGFPmut (see Materials and Methods) RNA together with XGRIP2.1 specific MOs and grown to tailbud stage (st. 30). (B) Total protein extracts of injected embryos were subjected to Western blotting. The recombinant mycGFP reporter protein was no longer detected at high concentrations of either MO1 or MO2. (C, D) mycGFP reporter protein expression analyzed by fluorescence microscopy. (a-c) normal light, (a’-c’) UV-light. Two-cell stage embryos were coinjected with 0.2 ng 5’UTRxGRIPmycGFPmut1 RNA and 2 pmol MO1 (C, a, a’), 0.2 ng 5’UTRXGRIP2.1mycGFP RNA and 0.2 pmol MO1 (C, b, b’), 0.2 ng 5’UTRXGRIP2.1mycGFP RNA and 2 pmol MO1 (C, c, c’), 0.2 ng 5’UTRXGRIP2.1mycGFPmut2 RNA and 2 pmol MO2 (D, a, a’), 0.2 ng 5’UTRXGRIP2.1mycGFP RNA and 0.2 pmol MO2 (D, b, b’), or 0.2 ng 5’UTRXGRIP2.1mycGFP RNA and 1 pmol MO2 (D, c, c’).
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Supplementary Fig. 3.
Fig. 3. Examples of phenotypes induced by overexpression of PDZ 23. Embryos were injected vegetally into both blastomeres at the 2-cell stage, grown to stage 31-32 and subjected to WMISH. (A) Control embryos exhibit normal average PGC numbers and positioning along the A/P axis. (B, C) Embryos injected with 2 fmol (B) or 4 fmol (C) of PDZ 23 RNA exhibit reduced average PGC numbers and PGC mislocalization along the A/P axis.
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Supplementary Fig. 4.
Fig. 4. Rescue of PDZ 23 induced effects upon coinjection of the wtORF. Embryos were injected with 2 fmol of PDZ 23 RNA alone or together with 0.2 ng XGRIP ORF vegetally into both blastomeres at the 2-cell stage, grown to stage 32, subjected for the whole-mount in situ hybridization and cleared with benzyl benzoate/ benzyl alcohol (2:1). (A) Embryos coinjected with PDZ 23 and XGRIP ORF exhibit a normal average number and regular positioning of PGCs along the A/P axis. (B) Embryos injected with PDZ 23 alone exhibit reduced average PGC numbers, as well as PGC mislocalization along the A/P axis (arrows).
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grip2.1 (glutamate receptor interacting protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 2, vegetal view.
Expression is in the germ plasm of the vegetal hemisphere
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Fig. 2.
XGRIP2.1 knockdown results in reduced average PGC numbers. (A) Schematic illustration of morpholino oligonucleotide target sites. 5′UTR and ORF of XGRIP2.1 cDNA are marked as blue box and grey arrow, respectively. (B–N) Embryos were injected vegetally into both blastomeres at the 2-cell stage. (B) XGRIP2.1 MO1 and MO2 inhibit translation of XGRIP2.1 in vivo. Embryos were injected with XGRIP2.1 5′UTR_ORF RNA with or without coinjection of XGRIP2.1 specific MOs or the control morpholino and grown to the tailbud stage 32. Total protein extracts of injected embryos were subjected to Western blotting. XGRIP2.1 protein was no longer detected at high concentrations of either MO1 or MO2. (C, D) Injection of either XGRIP2.1 MO results in a dose-dependent decrease of the average PGC number. The average PGC number was calculated from three independent experiments (see Materials and methods). The control score was in the range of 10 to 25 PGCs per embryo for the MO1 series (control in panel C) and 10 to 16 PGCs per embryo for the MO2 series (control in panel D), n—number of analyzed embryos. (E–G) Representative examples of embryos injected with the control morpholino, XGRIP2.1 MO1 or with XGRIP2.1 MO2, respectively. (H–N) Reduced PGC number in MO2 injected embryos as evidenced by use of different PGC markers. Embryos were injected with 1 pmol of CO MO or 0.2 pmol of XGRIP2.1 MO2, cultivated to stage 31/32 and subjected to WMISH. (H) Average PGC number in control and MO2 injected embryos. The result was calculated as an average of three independent injection series, 60 embryos were analyzed for each treatment (see Materials and methods). (I–N) Representative examples of embryos injected with CO MO (I, K, M) or XGRIP2.1 MO2 (J, L, N) and stained for Xdazl (I, J), XDeadSouth (K, L), XGRIP2.1 (M, N). ∗—p(t) < 0.05.
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Fig. 3.
Positioning of PGCs along the A/P axis in control and XGRIP2.1 morphant embryos. (A–H) Representative examples of uninjected control embryos exhibiting normal PGC positioning between stage 24 and stage 32. PGCs were stained for Xpat and somites for XMyoD by WMISH. (A–G) Embryos were cleared with benzyl benzoate/benzyl alcohol. (H) PGC migration profile of uninjected control embryos (stage 24–33). (J) The PGC distribution profile of embryos at stage 32 injected vegetally into both blastomeres at the 2-cell stage with 0.2 pmol of MO2 XGRIP2.1 or CO MO. Average PGC numbers at each somite position were calculated from results of 3 independent experiments. A total of 60 embryos were analyzed for each stage of development or treatment.
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Fig. 4.
Misexpression of the putative dominant negative XGRIP2.1 fragment PDZ 23 impairs normal PGC development and migration. (A, B, D) Embryos were injected vegetally into both blastomeres of the 2-cell stage. (A) Injection of synthetic mRNAs (2 fmol each) encoding for a series of putative dominant negative protein fragments of XGRIP2.1 results in a reduced average PGC number and mislocalization of PGCs. ∗—p(t) < 0.1 for average numbers of mislocalized PGCs and p(t) < 0.05 for average PGC numbers. (B) The PGC migration profile of uninjected control embryos (stage 32–33) (dark blue curve) and embryos injected with PDZ 23 RNA (2 fmol) alone (red curve) or together with XGRIP2.1 ORF RNA (0.2 ng) (green curve). (C) An uninjected control embryo (stage 33) illustrating the borders (somite numbers, white lines) of normal PGC positioning along the A/P axis. (D) An extreme example of an embryo injected with 2 fmol of PDZ 23 RNA exhibiting mislocalization of PGCs. (E) A representative example of an embryo injected with 2 fmol of PDZ 23 RNA exhibiting mislocalization of PGCs. N—number of independent injections, n—number of embryos analyzed in each experiment.
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Fig. 5.
Dominant negative activity of XGRIP2.1 PRZ 23 requires structural integrity of the predicted cargo binding domain. (A) Alignment of PDZ domains 2 and 3 of XGRIP2.1 with the corresponding domains of Rattus norvegicus GRIP1/GRIP2. Identical amino acids are marked as shaded boxes. The red box depicts the internal deletion introduced into the XGRIP2.1 PDZ 23 del construct; three amino acid changes introduced into PDZ 23 SRSmut are shown in orange. (B) The PGC migration profile of uninjected control embryos (dark blue curve), embryos injected with 2 fmol of PDZ 23 (red curve), PDZ 23 del (yellow curve) or PDZ 23 SRSmut (light green curve) mRNA. n—number of embryos analyzed. (C) Injection of 2 fmol of the PDZ 23 RNA increases the average number of mislocalized PGCs, calculated as percentage of the total PGC number from three independent experiments (see Materials and methods). 60 embryos were analyzed in total for each kind of treatment. n—number of PGCs scored. (B–C) Embryos were injected vegetally into both blastomeres at the 2-cell stage. n—number of PGCs scored, N—number of independent injections.
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Fig. 6. Coinjection of wtORF XGRIP2.1 can rescue the average PGC number and anteroposterior positioning of PGCs induced by 0.2 pmol of MO2 XGRIP2.1. Mutations within PDZs 2 or 3 abolish the rescuing activity. Embryos were injected vegetally into both blastomeres at the 2-cell stage. n—number of PGCs scored, N—number of independent injections. ∗—p(t) < 0.05.
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