Polarization of PCP protein localization in the migratory environment.

<p>(A-A”) Dorsal view with anterior to the top of a 24 hpf wild type floorplate at the level of r4 co-immunostained with anti-Vangl2 (green) and anti-ZO-1 (magenta), a marker of apical tight junctions. The boxed regions in A are examples of anterior-posterior membranes (AP) (61–90° from AP axis), intermediate membranes (I) (31–60° from AP axis) and lateral membranes (L) (0–30° from AP axis). Arrows in A” indicate enrichment of anti-Vangl2 labeling at AP membranes. (B) Quantitation of fluorescent intensity of anti-Vangl2 labeling for AP, I and L membranes. N = 5 embryos, 192 membranes (57 L, 47 I, 88, AP). Graph represents data as mean ± SEM. *p = 0.018, ****p<0.0001; Significance was determined using a paired two-tail t-test with Welch’s correction. (C-D) Live lateral views of 48 hpf wild type floorplate cells at the level of the spinal cord with mosaic expression of GFP-Vangl2 (C) and Fzd3a-GFP (D). Anterior is to the left and dorsal/apical is up; white dots indicate the center of each expressing floorplate cell, arrows indicate anterior subapical membrane enrichment of GFP-Vangl2 (C) and posterior subapical enrichment of Fzd3a-GFP (D). (E-E”) Dorsal view of the apical surface of floorplate cells in a 48 hpf embryo expressing GFP-Vangl2 (green) and stained for ZO-1 (magenta) Anterior is to the left; white dots indicate the center of the expressing cell. Arrows in E” indicate anterior enrichment of GFP-Vangl2. (F-F”) <i>En face</i> view of the apical endfeet of neuroepitheilial cells in r4 of a 24 hpf embryo expressing GFP-Vangl2 (green) and stained for ZO-1 (magenta). Anterior is to the left; white dot indicates the center of the expressing cell. Arrow in F” indicates anterior enrichment of GFP-Vangl2. N = 17 embryos, 23 cells. Scale bars: 5 μm.</p

Vangl2 and Fzd3a have opposing cell-autonomous and non-cell-autonomous roles in modulating filopodial dynamics.

<p>(A-E) Time-lapse spinning-disc confocal series of donor-derived FBMNs in chimeric embryos at 24–30 hours post-fertilization (hpf). Transplant conditions are indicated on the left as donor→host. Colored arrows indicate individual filopodia at different time-points. Anterior is to the top and medial is to the right. Scale bar: 5 μm. (F) Quantitation of filopodial lifetime for donor-derived FBMNs. Each data point is an average of all the filopodial lifetimes for one FBMN. (G) Quantitation of the maximum filopodial length for donor-derived FBMNs. Each data point is the average maximum length for all the filopodia of one FBMN. WT→WT: N = 6 embryos, 11 neurons (3 in r4, 4 in r5, 4 in r6), 70 filopodia; <i>vangl2</i>^<i>-/-</i>→<i>vangl2</i>^<i>-/-</i>: N = 6 embryos, 9 neurons, 43 filopodia; <i>vangl2</i>^<i>-/-</i>→WT: N = 6 embryos, 8 neurons, 44 filopodia; <i>fzd3a</i>^<i>-/-</i>→WT: N = 7 embryos, 7 neurons, 73 filopodia; WT→ <i>vangl2</i>^<i>-/-</i>: N = 8 embryos, 10 neurons, 152 filopodia; WT→ <i>fzd3a</i>^<i>-/-</i>: N = 6 embryos, 10 neurons, 65 filopodia. Graphs represent data as mean ± SEM. *p<0.05, **p<0.01 compared to WT→WT control; n.s., not significant. Significance was determined using an unpaired, two-tail t-test with Welch’s correction.</p

GFP-Vangl2 is enriched to the tip of retracting FBMN filopodia.

<p>(A) Live confocal image of a single GFP-Vangl2 expressing FBMN (green) in a <i>Tg(isl1</i>:<i>mRFP)</i> (magenta) 24 hpf embryo. Scale bar: 10 μm. (A’,A”) Magnified views of the boxed region in A of the individual channels, GFP-Vangl2 and <i>Tg(isl1</i>:<i>mRFP)</i> respectively, at the time points indicated. The arrow in E’ indicates enrichment of GFP-Vangl2 at the filopodial tip. Scale bar: 10 μm. (B) Quantitation of filopodia tip/base mean fluorescent intensity ratio for mRFP and GFP-Vangl2 at the time-point before and during GFP-Vangl2 enrichment. Before enrichment the mean fluorescent intensity ratio of GFP and mRFP at the filopodia tip versus the filopodia base is approximately 1 (N = 9 filopodia). During the enrichment event this ratio for GFP-Vangl2 is 1.31 while the ratio remains close to 1 for mRFP (N = 12 filopodia). (C) Plot showing the change in filopodial length over time for 10 filopodia. The stars indicate the time-point that GFP-Vangl2 is enriched at each filopodium tip. The black trace corresponds to the filopodium in A’,A”. Graph represents data as mean ± SEM. ***p<0.001; Significance was determined using an unpaired, two-tail t-test with Welch’s correction.</p

Floorplate PCP is neither required nor sufficient for FBMN migration but can support the migration of WT FBMNs.

<p>Confocal images showing dorsal views of 48 hpf hindbrains with anterior to the top. (A,B) <i>Tg(shh</i>:<i>Gal4)</i>-driven expression of <i>Tg(UAS</i>:<i>Xdd1-GFP)</i> (A) and <i>Tg(UAS</i>:<i>Fzd3aΔC-GFP)</i> (B) does not disrupt FBMN (magenta) migration. N = 13 Xdd1-GFP expressing embryos and 26 Fzd3aΔC-GFP expressing embryos. (C,D) <i>Tg(shh</i>:<i>Gal4)</i>-driven floorplate expression of GFP-Vangl2 (blue) in the floorplate of a wild type sibling does not disrupt FBMN migration (magenta) (C) and does not rescue migration in a <i>vangl2</i> mutant (D). N = 24 <i>vangl2</i> mutants with GFP-Vangl2 expression in the r4 floorplate, 14 with 5 or more expressing floorplate cells in r4. (C’,D’) Boxed regions from panels C and D respectively, showing a single z-plane where GFP-Vangl2 (blue) is expressed broadly in floorplate cells whose membranes are marked with the Zn5 antibody (yellow) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005934#pgen.1005934.ref069" target="_blank">69</a>]. (E,F) Genetic chimeras. Cascade blue-dextran marks all donor-derived cells (blue), <i>Tg(isl1</i>:<i>mRFP)</i> marks host FBMNs (magenta in E) and <i>Tg(isl1</i>:<i>GFP)</i> marks wild type donor-derived FBMNs (green in F). (E) The presence of wild type floorplate cells (blue) in a <i>vangl2</i> mutant host embryo does not rescue the migration of host FBMNs. N = 16 embryos with extensive contribution of WT cells to the floorplate. (F) The presence of wild type floorplate cells (blue) in a <i>vangl2</i> mutant can, however, support the migration of co-transplanted wild type donor derived FBMNs (green, arrows). N = 8 embryos with migrated donor-derived FBMNs/22 embryos with donor-derived FBMNs; N = 76 migrated FBMNs/383 total donor-derived FBMNs. (E’,F’) Single Z-planes of the boxed regions from panels E and F respectively, show that donor-derived cells (blue) are in the Zn-5-positive floorplate (yellow). Scale bars: 50 μm, 5μm in the insets.</p

Post-mitotic FBMNs require PCP signaling for migration.

<p>(A) Schematic of the late stage FBMN transplantation procedure in which a small number (1–5) of post-mitotic, pre-migratory FBMNs are moved from r4 of a <i>Tg(isl1</i>:<i>GFP)</i> donor into r4 of a stage-matched, 16 hpf <i>Tg(isl1</i>:<i>mRFP)</i> host. (B, C) Live confocal images showing dorsal views of chimeras at 48 hpf with anterior to the top. Transplant conditions are indicated as donor→host. Cascade blue-dextran marks all donor-derived cells (blue), <i>Tg(isl1</i>:<i>mRFP)</i> marks host FBMNs (magenta) and <i>Tg(isl1</i>:<i>GFP)</i> marks donor-derived FBMNs (green). White arrows indicate donor-derived FBMNs at 48 hpf. (D) Quantitation of the percent of donor-derived FBMNs at 48 hpf that failed to migrate (r4), partially migrated (r5) or fully migrated (r6). Each histogram refers to the transplant condition in the image to its left and numbers indicate the number of FBMNs represented in each bar. WT→WT, N = 42 embryos, 174 FBMNs; WT→ <i>vangl2</i>^<i>-/-</i>, N = 16 embryos, 73 FBMNs. ***p<0.0001 compared to WT→WT control. Brackets indicate rhombomere location. Scale bar: 50μm.</p

PCP signaling is required within FBMNs and in their r4 environment.

<p>(A) Schematic showing a dorsal view of a 48 hours post fertilization (hpf) zebrafish hindbrain with anterior to the top. Facial Branchiomotor neurons (FBMNs) (green) migrate posteriorly from rhombomere (r) 4 to r6, leaving a trailing axon that exits from r4. The enhancer element <i>islet-1</i> (<i>isl1</i>) CREST drives expression in branchiomotor neurons (green); the <i>hoxb1a</i> element drives expression in r4 (light blue); <i>egr2b</i> drives expression in r3 and r5 (yellow) and <i>shh</i> drives expression in the floorplate (purple). (B,C, G-L) Live or (H) fixed confocal images showing dorsal views of the hindbrain of 48 hpf zebrafish embryos with anterior to the top. Brackets mark rhombomere (r) position. Scale Bar: 50μm (B) <i>Tg(isl1</i>:<i>GFP)</i> expression in a wild type embryo at 48 hpf. (C) <i>Tg(isl1</i>:<i>Dvl-DEP-GFP)</i> embryo with unmigrated Dvl-DEP-GFP-expressing FBMNs in r4. (D,E) Dorsal view of E12.5 mouse hindbrains with FBMNs (magenta) labeled with anti-Isl1 antibody. Dotted lines indicate the length of facial motor nucleus. Scale Bar: 100μm (D) Migrating FBMNs in <i>Vangl2</i>^{<i>LoxP/LoxP</i>} control embryos. N = 6 embryos. (E) Blocked FBMNs in <i>Vangl2</i>^{<i>LoxP/LoxP</i>};<i>Isl1</i>^<i>Cre</i> embryos. N = 9 embryos. (F) Quantitation of FBMN migration stream length in <i>Vangl2</i>^{<i>LoxP/LoxP</i>} control embryos and <i>Vangl2</i>^{<i>LoxP/LoxP</i>};<i>Isl1</i>^<i>Cre</i> embryos. ***p = 0.0003. Significance was determined using an unpaired, two-tail t-test. (G-L) FBMNs (magenta) are either expressing <i>Tg(isl1</i>:<i>mRFP)</i>(G,I-L) or are stained with anti-Isl1 (H). (G,H) <i>Tg(egr2b</i>:<i>KalTA4)</i>-driven expression of <i>Tg(UAS</i>:<i>Xdd1-GFP)</i> (G) and <i>Tg(UAS</i>:<i>Fzd3aΔC-GFP)</i> (H), throughout r3 and r5 does not block FBMN migration. (I,J) <i>Tg(hoxb1a</i>:<i>Gal4)</i>-driven expression of <i>Tg(UAS</i>:<i>Xdd1-GFP)</i> (I) and <i>Tg(UAS</i>:<i>Fzd3aΔC-GFP)</i> (J), throughout r4 blocks FBMN migration out of r4. (K,L) Chimeric embryos with transplant conditions indicated as donor→ host. Cascade blue-dextran marks all donor-derived cells (blue) and <i>Tg(isl-1</i>:<i>mRFP)</i> marks all donor-derived FBMNs (magenta). (K) Wild type donor-derived FBMNs migrate normally in a non-transgenic control host. N = 37 embryos, 378 FBMNs. (L) Wild type donor-derived FBMNs fail to migrate out of r4 that is expressing <i>Tg(UAS</i>:<i>Xdd1-GFP)</i>. N = 26 embryos, 190 FBMNs. Inset: same image without the magenta channel showing that donor-derived FBMNs (blue, circled) are not themselves expressing Xdd1-GFP (green). (M) Histograms indicate the percent of donor-derived FBMNs at 48 hpf that failed to migrate (r4), migrated partially (r5) or migrated fully (r6). Each histogram corresponds to the chimeric condition in the image to its left and numbers indicate the number of FBMNs represented in each bar.</p

Model of PCP regulation of directed neuron migration.

<p>Based on the filopodial dynamics and migratory behaviors of FBMNs we observed in genetic chimeras, and the localization of Vangl2 and Fzd3a we observed in FBMNs and the cells of their migratory environment, we suggest a model in which antagonistic interactions between Vangl2 and Fzd3a mediate the observed effects on FBMN filopodial dynamics and through them, directional neuron migration. Within FBMNs, Vangl2 (green) localizes to filopodial tips and destabilizes them while Fzd3a (magenta) has the opposite, stabilizing effect. In the planar-polarized cells of the migratory environment Vangl2 serves to stabilize filopodia while Fzd3a destabilizes them. In light of the known intracellular and intercellular interactions between Vangl and Fzd that underlie epithelial planar polarization, we hypothesize that interactions between Fzd3a and Vangl2 complexes destabilize one another intracellularly while they promote one another’s effects on the actin cytoskeleton when they interact across cell membranes. Whether these interactions provide <i>directional</i> cues for migration remains to be discovered.</p