XB-IMG-158548
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Fig. 5. Requirements for cell sorting from mixed aggregates. a Diagram of the assay: mixed aggregates composed of unlabelled cells and mGFP-expressing cells were fixed and cryosectioned. GFP-expressing cells were detected with an anti-GFP antibody (green) and nuclei were counterstained with Hoechst (red). b–g Representative examples, with detailed views in right panels. b Aggregates of unlabelled and labelled IM cells, which distributed randomly. c Positive control: GFP-labelled IM cells sorted efficiently from unlabelled ectoderm, forming sharply delimited groups. d–f Control IM cells were mixed with IM cells manipulated as follows: d C-cadherin overexpression; e expression of constitutively active RhoA; f EphrinB1+,3 expression; g substitution of mesodermal ephrinB2 and EphA4 (eB2MO and A4MO) with ectodermal ephrinB3 (eB3) and EphB4 (B4). Scale bars, main panel 200 µm, enlargement 100 µm. h Quantification of cell clustering, using a relative index of dispersion (rID). Clustering is defined as departure from a random distribution (index of 1). Box plot as above. The number of aggregates is indicated on top. One-sided Mann–Whitney test was used for statistical comparison. Colour coded asterisks as follows: Red, comparison to control IM; dark and pale purple, comparison to ephrinB1 + 3 expression and to ephrin/Eph substitution (eB3 + B4 + eB2MO + A4MO). Except for myosin depletion (MHCMO) and cadherin replacement (CcadMO + Ecad/Ncad), all manipulations led to some significant degree of clustering compared to control non-manipulated IM. Ephrins/Eph substitution led to the highest degree of clustering. i Quantification of the relative length of heterotypic interface (rLHI), defined as the ratio between the total length of heterotypic contacts and the theoretical minimal interface, calculated as the perimeter of a circle encompassing the total area of the labelled population (Supplementary Fig. 6). Conditions of ephrin ectopic expression alone or in combination with EphA4 depletion led to a significant shortening of rLHI. Separation was further improved by substitution of mesodermal with ectodermal ephrin/Eph. None of the other conditions significantly departed from the negative control aggregates of non-manipulated IM. Plot, statistical test and colour code as in h
Image published in: Canty L et al. (2017) © The Author(s) 2017. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |