(A) and (B) The tissue surface tensiometer (TST) and calculation of surface tension. An aggregate (A), placed between two parallel plates, is deformed by moving the plates closer together at time zero (B) (i). The upper (UCP) and lower compression plate (LCP) are not to scale and are magnified for better illustration (panel A). The UCP is connected to an electrobalance (B) through a nickel-chromium wire (NCW). The balance ensures that the UCP’s position is kept constant, i.e., the deformation initiated on the aggregate by moving the LCP up (at t=0) is kept constant over time, and the change in force is recorded (B) (ii). For a detailed description of the working principle, see Materials and Methods. From the geometry of the aggregate (B) (i) and the force at equilibrium, i.e., the force plateau in panel (B) (ii), the surface tension can be calculated by application of the Young–Laplace equation (B) (iii). R₁ and R₂ are the two primary radii of curvature, at the aggregate’s equator and in a plane through its axis of symmetry, respectively. R₃ is the radius of the contact circle with either compression plate. H is the distance between upper and lower compression plates (from Foty et al., 1994). (B) (iv) shows a zebrafish aggregate before compression (top), under compression (middle), and 1 sec after release from a 1.5 h-compression (bottom). The aggregate remains flattened right after release and only rounds up again slowly after force removal. (C) Surface tension is independent of aggregate size. (C) shows that surface tension values are independent of the size of the aggregate (volume) for the mesendodermal tissue. Data points are in green, and the mean surface tension is drawn as a dashed blue line. r denotes the correlation coefficient, whose low negative value shows that there exists a negligible negative correlation between aggregate volume and surface tension. This confirms that the calculated aggregate surface tension is a material property. (D) Surface tension values of the different zebrafish tissues. Error bars represent standard error of the mean σ values. Sample numbers for MZoep, Lefty, E-cadMO, and Cyclops represent the number of compressions performed for each data set and are 35, 38, 39, and 35, respectively. Statistical analysis was by ANOVA and Newman-Keul’s multiple comparisons test. Statistical difference (p<0.001) in σ-values was found between MZoep and MZoep+E-cadMO, MZoep and Cyclops, Lefty and MZoep+E-cadMO, and Lefty and Cyclops, respectively.

Time-dependent sorting (A,B) and envelopment (C,D) assays of MZoep (ectoderm, red) and Cyclops (mesendoderm, green) cells after 2.5 h (A,C) and 16 h (B,D) in hanging drop culture. Irrespective of whether tissues were initially mixed as single cells or as fused aggregates, the system reached a stable state with the ectoderm occupying the internal position relative to the mesendoderm. Intermixed Cyclops (red) and Cyclops (green) mesendoderm cells after 2.5 h (E) and 16 h (F) in culture. Here, no sorting occurs. Scale bar=150 μm.

Sorting (A,B) and envelopment (C,D) assays of control MZoep ectoderm (red) and Cyclops mesendoderm (green) (A,C) and E-cadherin MO-injected MZoep (red) and Cyclops mesendoderm (green) (B,D) after 16 h. In A and C, ectoderm is enveloped by mesendoderm, whereas in B and D, mesendoderm is enveloped by ectoderm+E-cadherin morpholino. The scale bar=150 μm.

(A) Sketch of the zebrafish shield region with different cell types: yolk syncytial layer (YSL), axial hypoblast (axial mesendodermal progenitors), epiblast (ectodermal progenitors), and epithelial enveloping layer (EVL). (B) The shield as a 2D-rectangular object. The axes are denoted x,y. We assume symmetry in z-direction and can thus treat the 3D problem in two dimensions. The arrows indicate the parabolic velocity profile in the x-direction. (C, D) Hanging drop experiments of endogenous zebrafish shield tissue. Just after excision, anterior axial mesendoderm progenitor (green) and ectoderm progenitor (red) tissues lie adjacent to one another (C). After several hours in hanging drop culture, the axial mesendoderm tended to adopt an external position relative to the ectoderm (D). Scale bar=100 μm.

The image analysis parameters P and S allow us to quantitatively distinguish various cell sorting configurations without any assumptions of cell-cell interaction. The horizontal axis denotes the normalized mathematical analog to a dipole moment, P, and the vertical axis denotes the normalized ratio of scattering amplitudes of red and green cells, S. Indicated are (mean±sem) of N=15 individual experiments for intermixed, enveloped, and separate states, and N=10 for the shield experiment. For the intermixed state image analysis was performed for MZoep ectoderm and Cyclops mesendoderm cells before sorting started or on mixtures of the same cell types. For the separated state a combination of ectoderm and mesendoderm aggregates prior to tissue engulfment was analyzed. The images analyzed for the enveloped state were ectoderm surrounded by mesendoderm. The shields analyzed were extracted from gsc-GFP embryos, which had been injected with rhodamine. The shield tissues assumed a final configuration similar to that of the enveloped state.