Chien YH et al. (2018), Mechanical Strain Determines Cilia Length, Moti...

XB-ART-54874

Dev Cell 2018 May 07;453:316-330.e4. doi: 10.1016/j.devcel.2018.04.007.

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Mechanical Strain Determines Cilia Length, Motility, and Planar Position in the Left-Right Organizer.

Chien YH , Srinivasan S , Keller R , Kintner C .

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The Xenopus left-right organizer (LRO) breaks symmetry along the left-right axis of the early embryo by producing and sensing directed ciliary flow as a patterning cue. To carry out this process, the LRO contains different ciliated cell types that vary in cilia length, whether they are motile or sensory, and how they position their cilia along the anterior-posterior (A-P) planar axis. Here, we show that these different cilia features are specified in the prospective LRO during gastrulation, based on anisotropic mechanical strain that is oriented along the A-P axis, and graded in levels along the medial-lateral axis. Strain instructs ciliated cell differentiation by acting on a mesodermal prepattern present at blastula stages, involving foxj1. We propose that differential strain is a graded, developmental cue, linking the establishment of an A-P planar axis to cilia length, motility, and planar location during formation of the Xenopus LRO.

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Species referenced: Xenopus laevis
Genes referenced: celsr1 foxj1.2 fzd3 mtor nodal2 vangl2

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	Figure 1 Strain, Polarized MTs, and PCP Components in the LRO (A–C) Diagrams of the vegetal (A) and dorsoposterior aspects (B and C) of the Xenopus embryo during gastrulation illustrate the development of patterns of strain in the IMZ, and the presumptive GRP, during gastrulation. See text for explanation. (D) After gastrulation, the LRO (boxed area in C, inverted and viewed from the inside) differentiates by forming flow producing cells medially (M; long motile cilia that reposition to the posterior cell edge), and flow sensing cells laterally (L; immotile short cilia that remain in a more central planar position). (E) The LWR of LRO cells at different stages are shown on a Tukey boxplot. (F) The LWR of cells in three LROs at stage 12 is plotted versus position along the M-L axis, combining data from both sides, showing the average for equivalent size bins, the 95% confidence range, and the best-fit curve (Table S2). (G and H) Polar plots summarizing MT orientation in medial (G) and lateral (H) LRO cells (Figures S2A and S2B, Table S1). (I) MT alignment within medial and lateral LRO cells, where each point represents the standard deviation from the mean for an individual cell (Table S1). (J) Plots summarizing the stable fraction of fz3-GFP at junctions oriented parallel (P) or orthogonal (O) to the A-P axis, in medial and lateral LRO cells (Table S1). (K and L) Cilia length (K) and planar positioning (L) are plotted versus cell position in the LRO at stage 18, as the average for equivalent size bins, the 95% confidence range, and the best-fit curve (Table S2). Planar positioning is measured as a distance from the geometric center (0) to the posterior cell edge (1) normalized to one. Error bars in (I) and (J) indicate the mean ± SD. Full statistics and sample size for these data are provided in Table S1.
	Figure 2 Ciliation Defects in Explants that Lack Strain (A–E) Dorsal explants (dotted line, A) cultured on FN (B) or placed together into a Keller sandwich (KS explants, D). Axial elongation is suppressed in FN explants (C, top view) but rescued in KS explants (E, top view). (F) LWR of LRO cells at a stage 12 equivalent in FN and KS explants summarized on a Tukey boxplot. (G–I) Basal body planar position in LRO cells in situ (G), or in an FN (H) or KS explant (I) at a stage 18 equivalent, plotted in relation to the geometric cell center (center) and the cell edge normalized to one. Plots are aligned to the A-P axis as indicated in KS explant (E) or FN explant (white line, C). Data from different embryos or explants are color coded. (J) Plots of cilia length at the midline of the explants or the LRO (mean ± SD). Full statistics and sample sizes are provided in Table S1.
	Opens large image Figure 3 Strain Promotes Planar Axis Formation in LRO Explants (A–F) LRO explants isolated as indicated (A) were left unstrained (B) or gradually strained (C) by aspiration (white arrow) into a capillary (50 Pa, 3 hr). At a stage 12 equivalent, cell elongation (LWR) and orientation were scored and shown on a Tukey plot (D), or Rose plot (E and F), respectively. The number of cells (explants) scored is indicated. Rose plots indicate the fraction of cells scored that align to the x axis (set to the long axis of the capillary in strained explants or to the mean long axis in unstrained explants), versus orthogonal, y axis. (G–I) The stable fraction of fzd3-GFP (G), vangl2-GFP (H), and celsr1-GFP (I) was measured using FRAP, at junctions in the LRO explants at a stage 12 equivalent, either unstrained or after 3 hr of aspiration (50 Pa). Each point represents a junction measured, randomly chosen in unstrained explants or chosen in strained explants based on whether they lie parallel (P) or orthogonal (O) to the strain axis. Error bars show the mean ± SD. Full statistics and sample size are presented in Table S1.
	Figure 4 Strain Promotes Long Motile Cilia Formation in LRO Explants (A and B) Confocal images of LRO explants at a stage 18 equivalent, either left unstrained (A) or strained for 3 hr using 50 Pa of negative pressure (B), visualizing cell boundaries (blue), basal bodies (green), and cilia (red). Scale bars, 5 μm. (C) Cilia length at a stage 18 equivalent in LRO explants either left unstrained or strained for 3 hr using relatively high (50 Pa) or low (20 Pa) levels of negative pressure, with the mean ± SD indicated. (D and E) Confocal images of representative cilia in strained (E) and unstrained LRO explants, expressing tektin2-GFP, and stained with the acetylated tubulin antibody. Arrows mark basal body location of tektin2-GFP. (F and G) Diagrams (F) illustrating two different strain applications that vary in strain duration (3 versus 1 hr), following by cilia length measurements (G) at a stage 18 equivalent. (H) Cilia length at a stage 18 equivalent in LRO explants that were strained for 3 hr with 50 Pa, with the mean ± SD indicated. Shown are plots of data from individual explants where cilia length is plotted versus cell position in the explant during strain, showing the average for equivalent size bins, the 95% confidence range, and the best-fit curve (Table S2). Full statistics and sample size are provided in Table S1.
	Figure 5 Strain Induces Cilia Positioning and a PCP Vector in a Consistent Planar Direction (A–E) LRO explants (A) were left unstrained (B and D) or strained for 3 hr using 50 Pa of negative pressure (C and E). Basal body position plotted as in Figure 2 with different colors used in these and other panels to represent data from different explants. Plots are oriented along the inside-to-outside axis of the capillary in the strained explants but randomly assigned in the unstrained explants. Examples of basal body positioning data from unstrained (D) and strained explants (E) where the red dot is the geometric center, the blue dot is the basal body, black is the cell edges, and the arrows denote the planar vector. (F–H) Planar positioning of cilia in the LRO in situ (F) shown in polar plots as above, based on data obtained for lateral (G) versus medial cells (H). (I) FRAP analysis of isolated, Fzd3-GFP expressing cells in LRO explants at a stage 12 equivalent. Inside and outside refer to measurements made at cell edges furthest and closest to opening of the capillary during aspiration, respectively. Each point represents a measurement at one edge, with the mean ± SD indicated. (J–Q) Marginal zone explants, centered either 45° (J–M) or 90° (N–Q) from the dorsal midline, were left unstrained (K and O) or strained (L and P) using the same conditions employed for LRO explants. At a stage 18 equivalent, cilia length (M and Q) and planar positioning (K, L, O, and P) were scored and plotted, as above. Full sample size and statistics are presented in Table S1.
	Figure 6 Foxj1 Is Required for Strain-Induced Cilia Differentiation (A–D) Cilia length (D) and planar position (B and C) were measured in the LRO of foxj1 Crispr mutants at stage 18 (A), in cells located laterally (B) or medially (C). (E–H) Cilia length (H) and planar position (F and G) were measured at a stage 18 equivalent in LRO explants isolated from foxj1 mutants (E) and left unstrained (F) or subjected to strain (G) for 3 hr using 50 Pa of negative pressure. Cilia length is shown with the mean ± SD indicated. Full statistics and sample sizes are provided in Table S1.
	Figure 7 Deconstructing Cilia Differentiation in Response to Strain (A–L) Ectodermal explants expressing foxj1 (A–D), nr2 (E–H), or both foxj1/nr2 (I–L) were left unstrained (B, F, and J) or strained for 3 hr using 50 Pa of negative pressure (C, G, and K). At a stage 18 equivalent, cilia length (D, H, and L) and planar positioning (B, C, F, G, J, and K) were scored as described in Figure 5. The axes in (B), (F) and (J) were assigned randomly for each individual explant, with data from a given explant color coded. Polar plots in (C), (G), and (K) are oriented relative to the inside and outside of the capillary during strain. Cilia length is shown with the mean ± SD indicated. Full statistics and sample sizes are provided in Table S1.

References [+] :

Antic, Planar cell polarity enables posterior localization of nodal cilia and left-right axis determination during mouse and Xenopus embryogenesis. 2010, Pubmed, Xenbase