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Fig. 1. xKIFI3B expression characteristics during oogenesis and embryogenesis. (A–G) xKIF13B mRNA was detected by whole mount in situ hybridization. During oogenesis, xKIF138 mRNA localizes to the tip of the vegetal cortex; A. early (stage II) oocytes; and B, late (stage III–IV) oocytes. At the 4-cell stage, xKIFI3B mRNA is enriched in the granular germ plasm islands at the vegetal pole (C, vegetal pole view). At gastrula stage, the transcript is detected in the region of the primordial germ cells (D, blastoporus view). During neurulation, xKIF13B mRNA starts to be expressed in the neural tube and in the cranial neural crest cells (E, anterior view, and F, dorsal view). At tailbud stage, xKIFI3B mRNA is expressed in PGCs (arrow), neural crest cells, brain, spinal cord and notochord (G, lateral view of the embryo with the head to the left).
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Fig. 2. Knockdown of xKIF13B results in PGC mislocalization. All embryos were stained for Xpat (PGC marker) and MyoD (somite marker). (A and B) Representative examples of embryos injected with 33.6 ng of control MO (A), or with 16.8 ng of xKIF13B M02 (B). Quantifications of the average number of mislocalized PGCs (C and E) and of PGC “spreading” (D and F) in control MO (C and D) and xKIF13B MO injected (E and F) embryos (see also Material and methods). (G) xKIF13B loss- and gain-of-function reduces the average PGC number and leads to PGC mislocalization. Control embryos were injected with 33.6 ng of control MO: xKIF13B morphants were injected with 16.8 ng of xKIF13B MO2; rescue was performed by co-injection of 16.8 ng of xKIF13B MO2 and 0.1 ng of the xKIF13B ORF_DE mRNA; for xKIF13B overexpression 0.1 or 1 ng of the xKIF13B ORF_DE mRNA per embryo was injected. For each independent injection 20 embryos per treatment were analyzed. Average PGC numbers were calculated in percentage relative to the control embryos set to 100% for each independent round of injections. PGC spreading represents the minimal ellipse covering the germ cells, normalized to the size of the embryo and represented in arbitrary units (a.u). PGC mislocalization was calculated as percentage of mislocalized (ectopic) PGCs relative to the total PGC number. PGCs were regarded as “mislocalized” when positioned beyond somites 5–11 along the A/P axis of an embryo. Results were averaged between at least three independent rounds of injections. Error bars represent the standard deviation of the mean, lines depict p < 0.05.
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Fig. 3. Modulation of xKIF13B expression affects morphology, dynamics of protrusion forrmation and PIP 3-mediated polarity in isolated PGCs. (A and B) PIP3 is enriched in the protrusions forrmed by PGCs (A and A′) confocal microscopy images of a migrating PGC in a living embryo at stage 31. Images were taken in two channels: GFP to visualize EGFP_GRPIPH_DE reporter protein as a read-out for the PIP3 signal (A) and RFPto visualize farnesylated RFP (mRFP) labeling the cell membrane (A′). The arrow depicts the PIP3 enrichment in the protrusion at the leading edge of the cell. (B–B′) Fluorescence microscopy images of a PGC isolated from stage 31–32 embryos. Images were taken in two channels: (B) for GFP, to visualize EGFP_GRPIPH_DE reporter protein as a read-out for the PIP3 signal, (B′) for RFP to visualize farnesylated RFP (mRFP), labeling the cell membrane. The merged image (B′) reveals enrichment of PIP3 in the protrusion. (C–E) Representative examples for membrane dynamics and PIP3 distribution in cultured PGCs upon modulation of xKIF13B activity. PGCs were isolated from embryos that had been injected with 0.2 ng of the EGFP_GRPIPH_DE mRNA alone (C), co-injected with 10 ng of xKIF13B MO1 (D and E), or co-injected with 0.5 ng of the xKIF13B ORF_DE mRNA (F). Images were selected from time-lapse movies taken for 5 mm with a 10 s per frame mode. Arrows indicate PIP3-enriched membrane extensions formed by PGCs. (G) xKIF13B localizes to the membrane of PGC protrusions. 80×-snapshots from the confocal time-lapse movie illustrate the intracellular localization of xKIF13B in an isolated PGC on fibronectin. Cells were isolated form tailbud stage embryos co-injected at two-cell stage with 0.2 ng of the EGFP_GRPIPH_DE and 0.3 ng of the dsRed_xKIF13B ORF_DE mRNA. Images were taken in two channels: GFP to visualize the PIP3 signal (left image) and RFP for xKIF13B (middle image). The arrow points to xKIF13B enrichment at the membrane of the PGC protrusion.
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Fig. 4. Inhibition of P13K signalling affects PGC morphology and dynamics of protrusion formation. (A–D) Representative examples of membrane dynamics and PIP3 distribution phenotypes exhibited by cultured PGCs. Cells were isolated from embryos injected with 0.2 ng of EGFP_GRPIPH_DE mRNA alone (A),or co-injected with either 0.3 ng of dnPI3K_DE mRNA (B), 0.3 ng of xPTEN_DE mRNA (C), or incubated in 100 μM LY294002 (D). Images were selected from time-lapse movies acquired for 5 min in the 10 s per frame mode. Arrows indicate PIP3-enriched membrane extensions formed by PGCs. (E–F) Quantification of the phenotypic effects observed upon modulation of xKIF13B activity and PIP3 signalling. Average PGC numbers and mislocalization were calculated as described in the legend to Fig. 2. (E) Overexpression of xPTEN or of dnPI3K reduces average PGC numbers and leads to increased PGC mislocalization. (F) Synergistic effects of low doses of xKIF13B MO and dnPI3K on PGC number and mislocalisation.
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Fig. 5. Protrusion polarity and directional migration of Xenopus PGCs in vitro. (A and B) Graphical representation of the PGC polarity assay. (A) Dissection scheme of a tailbud stage embryo. Dorso-anterior parts as indicated by the dotted line were used for the preparation of the dorsal protein extract (DEx), the remaining portion of the embryo was used either for the PGC isolation or for the preparation of the ventral protein extract (VEx). (B) Schematic illustration of the final set up for the PGC polarity assay (see Materials and methods for details). (C–F) Radar-graphs illustrating directionality of protrusion formation by PGCs cultured in the absence (C) or presence (D–F) of DEx. Cells were isolated from stage 30–31 embryos injected with 0.2 ng of EGFP_GRPIPH_DE mRNA alone (C and D), or co-injected with 16.8 ng of xKIF13B MO2 (E), or co-injected with 16.8 ng of xKIF13B MO2 together with 0.1 ng of the xKIF13B ORF_DE mRNA (F). At least 90 cells each were analyzed in three independent experiments. Other abbreviations: Up – up the gradient. Down – down the gradient. L/R – orthogonally to the gradient (to the left/right).
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Sppl. Fig 2. xKIF13B expression on the nervous system and migrating neural crest cells at tailbud stage. XKIF13B mRNA was detected by wmish. Dotted lines indictate section palnes shown in (q-d). Abbreviations: pe prosencephalon; op olfactory placode; ev eye vesicle; me mesencephalon; hb hindbrain ; ov otic vesicle; nc neural crest; sc spinal cord; no notochord.
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kif13b ( kinesin family member 13B ) gene expression in Xenopus laevis embryo, NF stage 28, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
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kif13b ( kinesin family member 13B ) gene expression in Xenopus laevis embryos, NF stage 3, assayed by in situ hybridization, vegetal view.
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At tailbud stage (NF stage 28), kif13b mRNA is expressed in primordial germ cells (arrow), as well as the neural crest cells, brain, spinal cord and notochord.
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At NF stage 3 (4-cell), kif13b mRNA is enriched in the granular germ plasm islands at the vegetal pole.
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