Panousopoulou E et al. (2013), The distribution of Dishevelled in convergently...

XB-ART-47504

Dev Biol 2013 Oct 15;3822:496-503. doi: 10.1016/j.ydbio.2013.07.012.

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The distribution of Dishevelled in convergently extending mesoderm.

Panousopoulou E , Tyson RA , Bretschneider T , Green JB .

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Convergent extension (CE) is a conserved morphogenetic movement that drives axial lengthening of the primary body axis and depends on the planar cell polarity (PCP) pathway. In Drosophila epithelia, a polarised subcellular accumulation of PCP core components, such as Dishevelled (Dvl) protein, is associated with PCP function. Dvl has long been thought to accumulate in the mediolateral protrusions in Xenopus chordamesoderm cells undergoing CE. Here we present a quantitative analysis of Dvl intracellular localisation in Xenopus chordamesoderm cells. We find that, surprisingly, accumulations previously observed at mediolateral protrusions of chordamesodermal cells are not protrusion-specific but reflect yolk-free cytoplasm and are quantitatively matched by the distribution of the cytoplasm-filling lineage marker dextran. However, separating cell cortex-associated from bulk Dvl signal reveals a statistical enrichment of Dvl in notochord-somite boundary-(NSB)-directed protrusions, which is dependent upon NSB proximity. Dvl puncta were also observed, but only upon elevated overexpression. These puncta showed no statistically significant spatial bias, in contrast to the strongly posteriorly-enriched GFP-Dvl puncta previously reported in zebrafish. We propose that Dvl distribution is more subtle and dynamic than previously appreciated and that in vertebrate mesoderm it reflects processes other than protrusion as such.

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Species referenced: Xenopus
Genes referenced: ctnnb1 dvl2 klf6 myc
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	Fig. 1. Dvl cytoplasmic distribution in CE chordamesoderm cells is not polarised and is similar to that of dextran. (a) Stage 12.5 embryos were optically sectioned horizontally with respect to the anteroposterior axis, somewhat oblique to the equatorial plane of the gastrula embryo. The NSB runs as two near-parallel lines defining the notochord (Nt) in the middle from somatic mesoderm (S) on either side. All NSB directed ends (asterisks) were grouped versus the anti-NSB directed, anterior (A) and posterior (P). (E) is ectoderm, (M) is mesoderm. (b) A horizontal confocal image of a stage 12.5 embryo showing mosaic expression of myc-Dvl (green cells). beta-catenin staining reveals cellular outlines and the NSB. ((c)â€“(e)) Colocalisation of Dvl and rhodamineâ€“dextran in a bipolar cell. (f) Bars represent the average fractional fluorescence measurements in the respective cell quadrants (excluding the nucleus) for Dvl and dextran in non-NSB-captured mediolateraly polarised cells at st.12.5. No statistically significant bias in the distribution of the Dvl/dextran ratio was found. Measurements are for 83 cells. Error bars are Â±2Ã—standard deviation.
	Fig. 4. Frequency distribution of GFP-Dvl puncta. (a) Cells expressing GFP-Dvl were segmented as described above (red line around the cell) and the segmentation used to define a cortical strip of 5 Î¼m (defined by outer red line and inner yellow line), which allows for puncta that are cortical, i.e. in very close proximity to the cell edge, to be counted. The cell domains were defined by the diagonals of a bounding rectangle (marked by white lines) and the length of each domain measured. The puncta identified and counted are marked by a red circle. (b) Puncta counts in each domain normalised to cell cortex length showed no statistically significant bias in their distribution. (c) The puncta count for each domain was normalised to the domain length, to account for different contour shapes between domains. No statistically significant bias was found between the four cell quadrants. Error bars are Â±2Ã— Standard error of the mean. ((c)â€“(e)). Controls for live imaging analysis of GFP-Dvl. (c) Single frame from a live stage 12.5 explant expressing 100 pg/embryo GFP-Dvl, showing a weak, diffuse fluorescence. (d) A stage 12.5 explant expressing 300 pg/embryo GFP-Dvl, fixed for 2 h at room temperature in MEMFA and imaged in 30% glycerol/PBS, showing bright cytoplasmic fluorescence and puncta. (e). Single frame from a live stage 12.5 explant expressing 250 pg/embryo of GFP, showing diffuse cytoplasmic fluorescence with no puncta. All injections were done at the 32-cell stage in blastomere C1.
	Supplementary Figure 1. Dishevelled and Dextran show the same type and amount of enrichment at the NSB. (a-c). A sample of an NSB-captured cell expressing Dvl ("a", green channel) and Dextran ("b", red channel). The white line in "c" (merged channels) marks the NSB. (d) Average, fractional fluorescence measurements of Dvl (green columns), Dextran (red columns) and the log of the Dvl/Dextran fluorescence ratio. Error bars are Â± 2 x standard error of the mean.
	Supplementary Figure 2. Distribution of Dishevelled in the sagittal plane throughout stages 11 to 12.5. Anterior is to the top, red staining is anti-b-catenin (Alexa 568 secondary antibody), green staining is anti-myc-Dvl (Alexa 488 secondary antibody). (a) a x20 magnification of a sagittal view of a stage 11 embryo. Scale bar is 100Î¼m. White dotted lines outline the boundaries between the ectoderm and mesoderm in stages 11.5 and 12 and between mesoderm and endoderm in stage 12.5. (d) A x 63 magnification of a sagittal view of a stage 11 embryo showing individual myc-Dvl expressing mesoderm cells. Scale bar is 100Î¼m. The dotted and dashed white lines outline the same tissue boundaries as in (a). (b) x20 magnification of a stage 12 embryo, where the white dotted and dashed lines depict the same tissue boundaries as in (a). (e) is a x63 magnification of a st12 embryo showing one ectodermal and three mesodermal myc-Dvl positive cells. (c) x20 of a late st12.5 embryo, where the dashed white lines depict the dorsal and ventral mesoderm boundaries and (f) and (g) are x63 magnifications of the same stage as (c), showing an example of non-boundary touching (f) and a boundary touching (g) myc-Dvl expressing cells. (h-m): Histogram bars represent the averaged fractional Dishevelled, Par-1A and GFP-CAAX measurements in each of the four cell measured domains (dorsal, ventral, anterior, posterior). Error bars are standard deviations intracellular distribution of Dishevelled (blue bars), Par- 1A (red bars) and GFP-CAAX (green bars) in sagittal view, from stage 11 to 12.5. (h) The distribution of Dvl, Par-1A and GFP-CAAX through stages 11, 11.5 and 12 has all been summarised, as they were very similar (whether cells were touching or not the dorsal boundary). For stage 12.5 cells that form part of the boundary (i, j) are presented separately than those that donâ€™t (k, l, m). Again, no significant enrichment in any cell domain or direction is observed for Dishevelled or Par-1A and the distribution of both proteins is highly nearly indistinguishable from GFP-CAAX.
	Fig. 2. Quantification of Dvl cortical localisation in CE chordamesoderm cells. (a)â(c) Plot of average, fractional fluorescence of Dvl and dextran in the four domains of the cell and the average ratio between Dvl and dextran signal. (a) Non-NSB captured cells bipolarly protruding, towards and away from the NSB (n=34). (b) Non-NSB captured cells monopolarly protruding, towards the NSB (n=31). (c) Non-NSB captured cells monopolarly protruding, away from the NSB (n=24). Error bars represent Â±2Ãstandard deviation. (d) Schematic diagram summarising the statistically significant accumulations of Dvl/Dex fluorescence ratio over the entire population of cells analysed. Both bipolarly protruding and NSB-directed protruding cells show Dvl/Dex accumulation in NSB-directed protrusions compared to other cell domains, as indicated. Asterisks represent significance levels for each domain-pairwise comparison, where (âââ) represents p<0.001, (ââ) is p<0.01 and (â) is p<0.05. The distances of cells to the NSB are not to scale.
	Fig. 3. Analysis of Dvl cortical localisation according to cellular location and distance from the NSB. Data from Fig. 2(a) and (b) are here shown broken down accoding to cellular location and distance (in cell diameters) from the notochordâsomite boundary. ((a)â(h)) Fractional mean fluorescence of Dvl and dextran and their ratio in each cell domain. ((a)â(d)). Bipolarly protruding ((a) and (c)) and NSB-directed protruding cells ((b) and (d)) within 2 cell diameters from the NSB showing similarly significant enrichment in NSB-directed protrusions of both prospective notochord cells ((a) and (b)) and prospective somite cells ((c) and (d)). Bipolar cells that are 3 or more cell diameters away from the NSB (e) show only a mild enrichment at the NSB directed cell ends and NSB-directed cells (f) show no significant enrichment. Both bipolar and NSB-directed cells 5 or more cell diameters away from the NSB show no enrichment of Dvl in any domain. Error bars are Â±2Ãstandard deviation. (i) Schematic diagram summarising the statistically significant accumulations of Dvl/Dex fluorescence ratio in sub-categories of cells as indicated. The thickeness of the green line represents the accumulation of Dvl/Dex fluorescence. Black brackets are representing the pairwise comparisons between cellular domains (using multiple post hoc tests with a Bonferroni correction). Asterisks represent significance levels for each domain-pairwise comparison, where (âââ) represents p<0.001, (ââ) is p<0.01 and (â) is p<0.05. The distances of cells to the NSB are not to scale.

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