Fig. 2

LPA Induces Cell Polarization to Stable-Bleb Cells In Vitro

(A) Percentage of polarized stable-bleb cells over time upon stimulation with 100 µM LPA.

(B) Percentage of polarized stable-bleb cells from blastula stage embryos (brown, n = 287) or 75% epiboly stage Tg(mezzo:eGFP) transgenic embryos with mesendodermal (green, n = 183) and ectodermal progenitors (blue, n = 152) after 10 min in the presence of 100 µM LPA (+LPA) or DMEM-F12 culture medium alone (Control [Ctrl] at blastula stage [n = 321], mesendodermal [n = 286], and ectodermal [n = 251]).

(C) Percentage of polarized stable-bleb cells cultured in DMEM-F12 medium (Ctrl, n = 272), 100 µM LPA (+LPA, n = 219) or 30 µM LPA supplemented with 10 µM Blebbistatin (+) (n = 167), 10 µM Blebbistatin () (n = 143), 10 µM Y-27632 (n = 149), 100 nM Latrunculin A (LatA, n = 134), and 500 nM Jasplakinolide (Jasplak) (n = 83).

(D) Recruitment of myosin II to the cortex of progenitor cells over time after application of 10 µM LPA by a micropipette. Data (black, mean ± SEM) and sigmoid fit function (red) with half time t_1/2 = 20 s (n = 30).

(E) Myosin II localization in progenitor cells cultured in DMEM-F12 medium alone (Ctrl), 1 µM or 10 µM LPA. Arrows mark blebs (yellow) and the cortex-depleted protrusion front of stable-bleb cells (red).

(F) Boxplot of relative cortical myosin II fluorescence intensities for increasing LPA levels in unpolarized progenitor cells (each n = 120).

(G) Cortical enrichment of Myl12.1-eGFP (n = 60), Lifeact-GFP (n = 60), and GPI-RFP (n = 33) in non-polarized progenitor cells treated with 10 µM LPA. Cortical fluorescence intensity values I_LPA are normalized to reference values without LPA stimulation I_Ctrl.

(H) Relative bleb sizes for increasing LPA levels (each n = 120).

(I and J) Relative bleb sizes (I) and relative cortical myosin II fluorescence intensities (J) under non-confined and confined conditions.

(K) Exemplary fluorescence images of non-polarized and polarized cells expressing Myl12.1 in non-confined and confined conditions.

(L) BF images of mesodermal progenitors cultured in non-confined and confined environments (top) and quantification of cellular protrusions (bottom).

(M) Time-lapse BF and fluorescence images of Myl12.1-eGFP localization in mesodermal progenitor cells cultured on a 2D substrate coated with Fibronectin upon LPA stimulation. n.s., not significant, p < 0.001. Boxplots show single cell data points (black) and median (red). All cells were obtained from embryos at sphere stage, despite (B), and cultured in suspension on a passivated substrate despite (L and M). n, number of cells. All scale bars represent 20 µm.

See also Figure S1, Movies S2 and S3, and Extended Experimental Procedures.