Houston DW , Elliott KL , Coppenrath K , Wlizla M , Horb ME .

R24OD030008 NIH HHS , R24 OD030008 NIH HHS , R01 GM083999 NIGMS NIH HHS , P40 OD010997 NIH HHS

             CRISPR-cas system

	Figure 1. Mutagenesis of wnt11b.L in Xenopus laevis. (A) Genealogy of Xla.wnt11bemNXR maternal-effect mutants. Females are represented by circles, males are represented by squares. The symbol in light grey indicates F0 mosaic; black indicates mutant at sgT1 site; dark grey indicates mutant at sgT2 site; full shading indicates homozygosity; half shading indicates heterozygosity. (B) Alignments of wild-type (wt) and mutant (–13del) nucleotide and predicted protein sequences. The CRISPR sgRNA target is in bold; protospacer adjacent motif is underlined. The vertical dashed line represents the exon 1-exon 2 boundary. Arrowheads indicate predicted signal peptide cleavage sites. (C) Embryos injected with wnt11b wild-type mRNA (wnt11b_wt; middle), wnt11b mutant mRNA (wnt11b_–13del; right) or uninjected (Uninj.; left). (D) Images of heterozygotes (+/Z; left) and maternal-zygotic wnt11b mutants (M/Z; right). Arrows indicate ectopic tailbuds. (E) Representative frames from time-lapse movies of a heterozygous control gastrula (+/Z; top) and a maternal-zygotic wnt11b gastrula (M/Z; bottom). Time stamps indicate time from the beginning of filming (h:min:s). Scale bars: 500 µm.
	Figure 2. Normal expression of wnt11b in mutants. (A) In situ hybridisation of nanos1, wnt11b and vegt expression in oocytes from wild-type and homozygous mutant females (stages I-II upper row in each set; stages IV lower rows; vegetal views). (B,C) Real-time RT-PCR analysis of wnt11b (B) and wnt11 (C) in wild-type and mutant oocytes (stage VI) and in heterozygotes and maternal-zygotic mutant embryos. Staging is by Nieuwkoop and Faber (1956). Green bars are the stage (st24) used for relative expression; mutant samples are in cyan. Scale bar: 250 µm.
	Figure 3. Laterality defects in wnt11b mutant embryos. (A,B) Ventral views of stage 46 control heterozygotes (+/Z), showing normal lateral asymmetry. The heart shape is diagrammed (lower right), showing the ventricle positioned on the left-side and the outflow tract oriented to the right. The intestines coil anticlockwise (white arrows); g.b., gallbladder (arrow). (C) Left-sided pitx2c expression at stage 30 in heterozygous controls (arrow). (C′) Absence of pitx2c expression on the right side of control embryos. (D,E) Ventral views of homozygous maternal-zygotic mutants (M/Z) at stage 46, showing examples of heterotaxy and situs inversus, respectively. The heart orientation is reversed in both examples, and the gut coils clockwise in E. Arrows are as above. (F-H′) pitx2c expression in M/Z mutants; (F,F′) examples of embryos with normal pitx2c expression patterns (left-sided expression only); (G,G′) examples lacking expression on left and right sides, respectively; (H,H′) examples of bilateral pitx2c expression in a subset of M/Z mutant embryos. Arrowhead in F indicates example of normal pitx2c expression in an axially truncated embryo. Scale bars: 500 µm.
	Figure 4. Delayed and reduced mesendodermal gene expression in maternal wnt11b mutants. (A,C,D,G,I,J,M,N) Phenotypes (A,G) and in situ hybridisation of mesendodermal markers (C, myod1; D, szl; I, wnt11b; J, wnt8a; M, sox17a; N, nodal3.1) in early neurula (A,C,D) and mid/early gastrulae (G,I,J,M,N) in controls (+/Z; left-hand panels). (B,E,F,H,K,L,O,P) Phenotypes (B,H) and in situ hybridisation of mesendodermal markers (E, myod1; F, szl; K, wnt11b; L, wnt8a; O, sox17a; P, nodal3.1) in early neurula stage (B,E,F) and mid/early gastrulae (H,K,L,O,P) in maternal mutants (M/Z; right-hand panels). Dorsal, posterior views (A-F); vegetal views (G-P, dorsal towards top). (Q-S) Real-time RT-PCR of myod1 and szl at stages 12/13 (Q), and sia1 (R) and szl (S) at stages 9 and 10.5. Green indicates the samples used for normalisation; maternal mutants are coloured cyan. Error bars represent the s.d. of two biological replicates (pools of three embryos); unpaired two-tailed t-test; **P<0.01, ***P<0.001, ****P<0.0001. Scale bars: 500 µm (in A for A,B,G,H; in C for C-F,I-P).
	Figure 5. Wnt11b does not regulate Lrp6 phosphorylation. (A) Immunoblotting of control oocytes and oocytes injected with mouse Lrp6 mRNA, untreated (oo.) or treated with progesterone (+prog.). The blot was reprobed using anti-pLrp6 (S1490) and anti-Lrp6 mAbs (anti-Lrp6 cocktail). Tubulin and di-phospho-ERK were used as loading controls. (B) Lrp6-injected oocytes were fertilised by host-transfer and blotted against anti-pLrp6 (S1490) and two anti-Lrp6 mAbs (anti-Lrp6 cocktail). A non-specific (non-spec.) band was used to assess equal loading. (C) Immunoblotting of wild-type (wt) and homozygous mutant female (−/−) oocytes, treated with progesterone, without or with prick-activation (+prick 60′). Relative migration (Mr) of molecular weight standards is on the left. Normalised relative quantification values for pLrp6 in panel C are shown below the blot.
	Figure 6. Wnt11b does not regulate Dvl puncta. (A-C) Control wild-type (wt) oocytes injected with dvl2-gfp and untreated (A), treated with progesterone (2 µM overnight; B) or treated and prick-activated for 60 min (+prick; C). (D-F) Homozygous mutant oocytes treated with or without progesterone and prick-activation. (G) Representative immunoblotting of Dvl2-GFP (anti-GFP) in wild-type (wt) and mutant (−/−) oocytes treated with or without progesterone and prick-activation. The loading control was β-tubulin. (G′) Normalised intensity values for anti-GFP bands (n=2 blots), plotted relative to injected wild-type oocytes (wt). The white bar in each box indicates the median value per group; top and bottom of the boxes indicate the 0.25 and 0.75 quantile limits, respectively. (H,I) Control oocytes injected with either rab5-gfp (H) or rab11-gfp (I) along with dvl2-mcherry. Scale bar: 10 µm.
	Figure 7. wnt11b is required for microtubule alignment during cortical rotation. (A-B′) Immunostaining of β-tubulin in fertilised wild-type (A,A′) or wnt11b mutant (B,B′) zygotes, fixed at 60 min postfertilisation (60′ p.f.). Shown are tiled images of the vegetal surface (A,B) and higher magnification confocal images (A′,B′). (C-D″) Frames from time-lapse movies of prick-activated wild-type (C) and wnt11b mutant (D) oocytes injected with eb3-gfp mRNA imaged at 60 min post-activation. (C′,D′) Consecutive frame averaging of microtubule motion (green 1-5>magenta 7-11). (C″,D″) Angle histograms of plus end directionality (degree). Circular statistics are listed; φ̅ (phi bar), mean angle, r, mean resultant vector length, Rayl-p, P-value of Rayleigh test for circular uniformity. (E,F) Box and whisker plots of mean r-value of prick-activated wild-type (E) and and wnt11b mutant (F) oocytes injected with eb3-gfp mRNA and imaged at the indicated time points post-activation (35-85 min). The dark bar in each box indicates the mean; the bottom and top edges of the box indicate the 0.25 and 0.75 quantiles, respectively; individual data points are shown. The P-values (one-way ANOVA) are at the top; the bracket indicates significance groups (Tukey's HSD criterion); n=3. n.s., not significant versus any group. (G) Correlation plots of r versus dynamic parameters and histograms of r. Magenta lines show the least squares fit line, the blue dotted line indicates the sample mean, correlation values are upper left (red colour indicates statistical significance; P<0.05). The rightmost panels show histograms of r values obtained for each sample. An r>3.0 is empirically significant in this context. Scale bars: 100 µm (A,A′, also for B,B′); 10 µm (C, also for C′,D,D′).
	Fig.S1. (A) Location and sequence informationfor sgRNAs against wnt11b.L. Genome browser view was obtained from Xenbase. (B) Expression ofwnt11b.L, wnt11.L and wnt11.S duringoogenesis and development. RNAseq expression data from Session et al. (2016) retrieved from Xenbase TPM=transcripts per million; oo=oocyte stage; NF=Nieuwkoop and Faber (1956) stage.
	Fig. S2. (A-C) Examples of an +Zwnt11b+I- embryo (A) and MZwnt11b-I- embryos (B-C) at the onset of gastrulation (NF stage 10-10 1/4), showing variation in timing of dorsal lip formation . (D-1)lmmunostaining of against F-actin, beta- tubulin and cytokeratin in heterozygous +/Z (D-F) or wnt11b mutant (G-1)gastrulae (stage 10). Equatorial images are shown (63x mag.), scale bars are 10 µm.
	Fig. S3. (A-D) Phenotypes of control (A. D) and maternal-effect wnt11b mutant embryos (B, C). (A'-D') Double immunostaining against somite (12/101; green) and notochord (Tor70; red) antigens. (A"-D") lmmunostaining against a neural-specific antigen (6F11 (NCAM); green). Arrows indicate ectopic tailbud-like structures
	Fig. S4. (A-H) In situ hybridization against nodal3.1 in maternally wildtype (wt) (A-B, E-F) and mutant wnt11b embryos (C-D, G-H) at stage 9 (A-D; dorsal views) and stage 10.5 (E-H; vegetal views, dorsal to top). (I) Real-time RT-PCR analysis of nodal3.1 at stage 9 and 10.5. The bar in green indicates the sample used for normalization of relative expression. Error bars represent standard deviation of two biological replicates. **** p < 0.0001 versus wildtype within stage group.
	Fig. S5. Heatmaps showing relative gene expression changes for selected germ layer and patterning markers at stage 9 (A) and stage 10.5 (B), stage 9 and 10.5 wnt genes (C) and top 16 differentially expressed genes across both stages (D). Values for each gene represent the differences from the mean value all samples, following regularized log2 transformation of counts. Replicates are biological replicates of three pooled embryos per replicate sample. Darker colors indicate relative downregulation; lighter colors indicate relative upregulation.
	Fig. S6. (A) Real-time RT-PCR analysis of ventral (sz and dorsal (sia1, noda/5, noda/3. 1) instage 1O embryos derived from wild type (wt) or wnt11b mutant (M/Z) oocytes fertilized by wild type sperm following host-transfer (three pooled oocytes/embryos per sample). Control heterozygous (+/Z) embryos were used for normalizationofrelative expression (green bar). (B-E) Representative phenotypes of control (+/Z; B, C) and wnt11b mutant (D, E) embryos obtained by host-transfer. Samples in B and D were left uninjected (uninj.);samples in (C) and (E) were injected as oocytes with 20 pg wnt11b mRNA. Dorsal views, anterior is up except top row in (D), where arrowheads indicate anterior.
	Fig. S7. (A) Representative phenoptyes of embryos derived from host-transferred oocytes left uninjected (uninj.) or injcted with low (A, 50 pg) or high doses (B, 250 pg) of mouse Lrp6 mRNA. d.a.i, dorsoanterior index (Kao and Elin- son, 1988) of the examples shown.
	Fig. S8. Additional microtubule tracking examples. (A-C)Representative single frames from time-lapse moviesofprick-activated control (A) or wnt11b-I- mutant oocytes (B,C) injected with eb3-gfp mRNA. (A'-C') Microtubule motion is depicted by averaging frames 1-5 andframes 7-11 andmerging pseudo-colored images green and magenta, respectively. (C-C") Angle histogram plots showing individual plusend track directionality (degree) per bin per two minute movie.An example of a wnt11b-l- mutant sample with lower directionality (r<0.1) is shown in C-C".Circular statistics are listed; cp (phi bar) = mean angle, r = mean resultant vector length, Rayl-p = p-valueof Rayleigh test for circular uniformity.
	Fig. S9. Correlation matrices and histograms (diagonals) of microtubule dynamic parameters in timelapse movies from wildtype (A) and mutant (B), prick-activated eggs imaged at 60 minutes postfertilization. Magenta lines show the least squares fit line, correlation values are upper left (red colour indicates statistical significance; P<0.05). Please note the differing scales between the two matrix plots and the scales in Fig. 7G; the bottom row of each panel is identical to the corresponding panels in Fig. 7G (except for scale and aesthetic adjustments).
	Fig. S10. Relative protein expression of Wnt11b protein during Xenopus laevis development. Data were visualized on xenbase.org and were derived from Peshkin et al. (2019). Nieuwkoop and Faber stages are shown on the x-axis, scaled to represent hours post-fertilization. The arrow indicates an increase in relative Wnt11b protein level in the fertilized egg. A larger increase is apparent during gastrulation.

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