Fig. 3: Basement membrane is heterogeneous before and during DVE migration.

a Schematics of a mid-E5.5 embryo with the perforated basement membrane (BM) in magenta that lies between the EPI and the VE. Ectoplacental cone (EPC) (gray), VE (white), ExE (cyan), EPI (yellow), and DVE (green) represent the major cell types at this stage. DVE migrates toward the left side of the embryo. b Maximum Z projection showing the EPI region of an E5.5 Cerl-GFP embryo imaged in toto with Airyscan2 super-resolution SR-8Y acquisition mode to map basement membrane organization. DVE are marked with Cerl-GFP in green and LAMININ immunofluorescence in magenta. The proximal (P)-Distal(D) axis of the embryo was shown. Scale bar: 20μm. c A zoomed-in image of an E5.5 embryo at the EPI and the embryonic VE (EmVE), showing the basement membrane in magenta and Cerl-GFP in green. The yellow arrow indicates the projected direction of DVE migration. The PD axis of the embryo was shown. Scale bar: 20 μm. d Flattened ImSAnE cylinder projection of the E5.5 embryo in (c), revealing the entire basement membrane, proximal(P) on top and distal(D) at the bottom, for visualizing the distinctive basement membrane organization at DVE versus non-DVE regions. e A fraction of the ImSAnE-flattened basement membrane is extracted and segmented with iLastik to quantify the percentage of perforations at the DVE side (Cerl-GFP⁺) versus the non-DVE side (Cerl-GFP^-) side. f Quantification of basement membrane asymmetry in airyscan2 super-resolution data of early E5.5 embryos. Measurements mathematically account for area distortion in the cartographic projection. A polar coordinate system centered around the distal tip was created for each embryo, and angles indicate the position along the circumference of the embryo. We define polar angle 0 as the region with the highest number of Cerl-GFP⁺ DVE cells. When aligned by position of the DVE of 10 individual embryos, there is an average 5% higher perforation on the side with the DVE. n = 10 embryos. one-sided t-test, p-value = 0.03(*). Shaded bands represent standard deviation. g Maximum Z projection images from airyscan2 stacks of representative E5.0-E5.5 embryos with basement membrane organization visualized with LAMININ in gray and DVE with CERL(CER1) in green. CERL protein was not detected at E5.0 and E5.25 and became visible at the distal tip of early-E5.5 and mid-E5.5 embryos (yellow asterisks). The basement membrane showed perforations from E5.0 to E5.5. Scale bars: 20 μm. h Plots of representative E5.0 and E5.25 embryos with their area-corrected basement membrane percentage as a function of the polar angle. A polar coordinate system centered around the distal tip was created for each embryo, with a polar angle measuring the position across the embryo circumference. Note that polar angles are not directly comparable across embryos as there is no expression to rotationally align multiple embryos. n = 1 and 1 embryo. i Quantification of the asymmetry of basement membrane perforations. EmVE was divided into seven circumferential regions and the differences of basement membrane perforation were measured with an asymmetry index, defined by the regions with maximum minus minimum levels of basement membrane perforation divided by the mean percentages of basement membrane perforation. An asymmetry index of 0 indicates symmetry. The percentage of perforations of the basement membrane are asymmetric before the stages of DVE migration and before the stage in which Cerl-GFP is expressed at the distal tip of the embryo. n = 9, 8, and 10 embryos. Error bars: standard deviation.