Fig. 1: Modulation of the level of basement membrane perforations affects DVE migration.

a Schematics of AP axis specification in mouse E5.0-E6.5 embryos. Three major cell types: the epiblast (EPI), extra-embryonic ectoderm (ExE), and the visceral endoderm (VE) are shown. A subset of VE, the distal visceral endoderm (DVE) cells begin to migrate collectively and unidirectionally toward the proximal region of the VE. At E6.5, anterior visceral endoderm (AVE) facilitates the localization of the primitive streak at the opposite end of the embryo. b Schematics of an E5.5 embryo with the perforated basement membrane (BM) (magenta) that lies between the EPI and the VE. c Control E5.5 embryos were stained with Laminin to check the architecture of the basement membrane. A yellow-dotted rectangle marks the basement membrane underneath the EPI. n = 10 embryos. Scale bars: 20 μm. d Snapshots of confocal time-lapse imaged control E5.5 embryos from Cerl-GFP transgenic mice. Brightfield and GFP (green) to track DVE migration. Scale bars: 20 μm. e The basement membrane underneath the EPI (yellow dotted line) in the collagenase-treated E5.5 embryo is depleted and thus does not exhibit a layer of Laminin with physical perforation as in control (c). n = 10 embryos. Scale bars: 20 μm. f Snapshots of confocal time-lapse imaged basement membrane-depleted Cerl-GFP transgenic E5.5 embryos. Brightfield and GFP (green) to track DVE migration. Scale bars: 20 μm. g, h DVE migrates faster in basement membrane-depleted embryos. Percentage migration is defined as the percentage displacement of the Cerl-positive cells along the proximal-distal path during one hour of in vitro culture. The position of the distal tip is defined as 0% and the ExE-EPI boundary is 100%. Each dot represents one embryo. Scale bars: 20 μm. Error bars: standard deviation. n = 10 and 13 embryos. Welch’s two-tailed t test, p-value = 0.002(**). i, j At E5.5, an embryo treated with 10 μM Batimastat shown in (j) is sufficient to reduce the frequency of basement membrane perforations compared to the control in (i). Scale bars:20 μm. k Snapshots from a time-lapse confocal live imaging of an E5.5 DMSO-treated control Cerl-GFP embryo that showed lateral migration once DVE reached the ExE-EPI boundary. n = 3 embryos. Scale bars:20 μm. l Snapshots from time-lapsed confocal imaging of an E5.5 Cerl-GFP (green) embryo cultured with 10 μM Batimastat. n = 8 embryos. Scale bars: 20μm. m Quantification of the DVE proximal migration speed defined by the percentage dislocation of the DVE cells toward the EPI-ExE boundary in control versus 10 μM Batimastat-treated embryos. Error bars: standard deviation. n = 3 and 8 embryos. Welch’s two-tailed t test, p-value < 0.0001(****). n Magnified image of the same embryo in (l), showing premature lateral migration before DVE reaches the EPI-ExE boundary. Lateral migrating Cerl-GFP positive cells can be seen around 9 h and are marked with yellow asterisks. Scale bar: 20 μm.