Extended Data Fig. 4: Measurement of cell division angles in the developing epithelium.

In 3D live imaging data, morphology of the developing epithelium was changing constantly. Accordingly, the basement membrane zone was also bending, but not flat. Therefore, we calculated the cell division orientation relative to the basement membrane zone in the developing epithelium three-dimensionally as shown here. a, We first marked the dividing cells in the placode and surrounding IFE in live imaging data, based on chromosome condensation. Examples of a perpendicular (left panels) and horizontal division (middle panels) relative to the basement membrane zone, which were pseudo-coloured in blue; the mitotic spindle axis is indicated by a cyan line connecting the daughter-cell nuclei. The top panels are a planar view images of the epithelium, and the bottom panels are sagittal view images of the epithelium. The placode region in the epithelium was distinguished by accumulation of Fucci-G1 signals accompanied by condensation and cell cycle arrest of the underlying dermal cells, as shown by the dashed circle in the right panel. Scale bars, 20 μm. b, To extract positional information of the basement membrane zone, we next obtained the surface of the developing epithelium based on expression of K14-H2B-eGFP using the surface rendering function in Imaris. A surface object in Imaris was constructed as a mesh object consisting of triangles and vertex normal vectors. Then, based on the direction of the normal vectors, we cut the surface of the opposite side of the basement membrane zone (outer surface of the epithelium) and substituted the remaining dermis-side surface for the basement membrane zone. c, We calculated the 3D orientation of cell division to the basement membrane zone using x–y–z axis coordinates of the mitotic spindle axis and basement membrane zone. In brief, we first found the closest vertex and its associated normal unit vector on the dermis-side surface by calculating the distance between the centre of a mitotic spindle and each vertex. The cell division angle to the basement membrane zone was then calculated from an inner product of the closest vertex normal unit vector and the unit vector corresponding to a mitotic spindle.