Oh D , Houston DW .

R01 GM083999 NIGMS NIH HHS

	Fig. 1. Germ plasm enrichment of Sybu mRNA and protein in oocytes and early embryos. (A-D) Representative images of whole-mount in situ hybridization on stage VI oocytes comparing expression of (A) trim36, (B) wnt11b, (C) pgat and (D) sybu. Vegetal views are shown. (E-J) Localization of Sybu-GFP to germ plasm. (E-G) Overlap of Sybu-GFP with clustered mitochondria (mito., MitoTracker) in pricked eggs 60-min post-activation (p.a.) and at 240-min post-activation (p.a.). Scale bars represent 7.5 µm.
	Fig. 2. Antisense oligo-mediated depletion of sybu. (A) Real-time RT-PCR analysis of total sybu levels following injection of as234. (B) Differential depletion of sybu homeologs by as234 and as6 oligos. (C) Representative embryos obtained from host-transfer experiments using Uninjected control oocytes (upper panel) or sybu-depleted oocytes (lower panel). In (A-B), bars represent the average of two experiments; errors bars represent standard errors.
	Fig. 3. Reduction of PGCs in sybu-depleted embryos. (A-C) Whole-mount in situ hybridization for pgat mRNA. (A) control uninjected embryo, (B) sybu-depleted embryo, (C) depleted embryo rescued with injected sybu mRNA (5 pg). (D) Scatter plots of PGC numbers in experimental groups corresponding to the adjacent panel. Dots represent individual in which PGCs were counted (sum of three separate host-transfer experiments); dark line represents the median; shaded areas correspond to the limits of the first and third quartiles (25th/75th percentiles) and whiskers (dotted line) extends out to 1.5x the interquartile range. P<0.001 by Kruskal-Wallis test; *** = P<1.0e-11 by Dunn's post hoc test w/ 2-sided Bonferroni adjustment.
	Fig. 4. Abnormal germ plasm aggregation in sybu-depleted embryos. (A-D) Representative embryos at the 4-cell stage, probed for pgat to identify germ plasm, vegetal views. (A) uninjected control embryo, (B) sybu-depleted embryo, using as234, (C) sybu-depleted embryo using as6, (D) sybu-depleted embryo (as234) rescued by injection of sybu mRNA. (A’-D’) Representative embryos similarly analyzed at the 32-cell stage.
	Fig. 5. Failure of peri-nuclear germ plasm localization in sybu-depleted embryos. (A-C’) DiOC6(3) staining in isolated blastomeres (Green; DAPI in blue). Embryos were stained at the 4-cell stage and dissociated at the early gastrula stage. (A, A’) control uninjected isolated vegetal blastomeres, (B, B’) sybu-depleted blastomeres, (C, C’) blastomeres rescued by sybu-mRNA injection. arrows = cortical mitochondrial/germ plasm enrichment; scale bar = 30 µm.
	Fig. 6. Time lapse imaging of germ plasm aggregation during early development in host-transfer fertilized eggs. (A) Representative time-lapse imaging of a zygote obtained by the host-transfer procedure, stained with DiOC6(3). Panels in (A) show selected frames at ~5-min intervals from Supplementary Video 1 (170 min time-lapse, frame rate = 20 s). In (A, C), elapsed time from in vitro fertilization is shown at the top of each frame (in h:min:sec); dotted lines indicate the lines used for kymograph profiles; brackets indicate areas of clustered germ plasm; scale bars = 85 µm. (B) Representative kymograph analysis of an uninjected control embryo. The approximate timing of post-fertilization events is shown on the right. The white arrow indicates the beginning of aggregation into larger areas at the tail end of SCW2a/b. The position arrow indicates distance from the vegetal pole (bottom left corner). (C) Representative time-lapse imaging of a sybu-depleted zygote obtained by the host-transfer procedure, stained with DiOC6(3) (labels as in A). Panels show selected frames at ~5-min intervals from Supplementary Video 2. (D) Representative kymograph analysis of a sybu-depleted embryo. Germ plasm is not disturbed by SCWs (arrowhead) and fails to form large continuous aggregates at the cleavage furrows.
	Fig. 7. Quantification of germ plasm aggregation in early development. (A-D) Representative images from time-lapse videos (Videos 1, 2) from uninjected and sybu-depleted embryos, showing frames analyzed at ~90 min (A, C) and ~170 min (B, D) after fertilization. Vegetal views. Thresholded areas were randomly pseudocolored to verify that they could be distinguished and measured as separate clusters. (E-F) Graphs showing mean aggregate size at the indicated time points; error bars indicate standard errors. N = 3 host-transferred eggs;>100 germ plasm aggregates (three experiments). The p-value from one-way ANOVA is indicated at the top. Scale bar in A-D = 85 µm.
	Fig. 8. Functional interaction of Sybu with kinesins Kif3b and Kif4a in germ plasm aggregation. (A) Model of the experimental design. Oocytes are stained with MitoTracker and imaged following prick-activation. (B-E) Representative images control eggs before and after aggregation (B, Uninjected (Un) egg; C, Un egg + prick activation (pk)) and of sybu-depleted and rescued prick-activated eggs (D, sybu- egg +pk; E, sybu- egg +pk + RNA). (F-G) Average germ plasm aggregate size in depleted and rescued groups (F) and in different combinations of sub-effective doses of oligos (G). The dark bar in each box indicates the median size per group, lower and upper edges of the colored boxes indicate 25th and 75th percentiles respectively, whiskers represent maximum and minimum adjacent values. Outliers (>1.5x interquartile range) are indicated by crosses (+). N = 20 oocytes,>500 total islands. ***P<0.001 by Kruskal-Wallis test/Dunn's post-hoc.
	Supplementary Fig. S1. In situ hybridization of sybu expression in (A) oocytes (stages I-IV upper row; stages V-VI lower row), (B) 4-cell embryos, and (C) gastrulae. Vegetal views.
	Supplementary Fig. S2. Antisense oligo-mediated depletion of kif3b and kif4a. (A) Real-time RT-PCR analysis of total kif3b and kif4a levels following injection of corresponding oligos. (B-C) Images of control uninjected (B) and kif3b-depleted gastrulae (C), vegetal views.
	Supplementary Fig. S3. Time lapse imaging of germ plasm aggregation during early development in kif3b-deplted, host-transfered eggs. (A) Representative time-lapse imaging stained with DiOC6(3). Panels in (A) show selected frames at ~5-min intervals from Supplementary Video 4 (170 min time-lapse, frame rate = 20 s). In (A), elapsed time from in vitro fertilization is shown at the top of each frame (in h: min:sec); the dotted line indicates the line used for the kymograph profile; scale bar = 85 µm. (B) Representative kymograph analysis of kif3b-depleted embryo. The approximate timing of post-fertilization events is shown on the right. The position arrow indicates distance from the vegetal pole (bottom left corner).

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