Figure 7. Rreb1-/- chimeras exhibit changes in ECM organization.
(A,C,D) Confocal images showing transverse cryosections of immunostained E7.5 chimeric embryos containing Rreb1+/+ or Rreb1-/- cells. (A) Confocal optical sections of Rreb1-/- chimeras. Cherry fluorescence is a constitutive lineage label marking the progeny of Rreb1-/-embryonic stem cells (ESCs) introduced into host embryos. Arrowheads mark ectopic SOX2 + cells derived from wild-type host cells (white) or from Rreb1-/- cells (magenta). Sb, 25 μm. (C) Confocal optical sections of Rreb1-/- chimeras. Arrowhead marks ectopic SOX2 + cells traversing a break in the basement membrane between the epiblast and outer visceral endoderm layer. Sb, 10 μm. (D) Confocal optical sections and maximum intensity projections (MIP) of Rreb1-/- chimeras. Upper and lower panels are sections taken from the same embryo, 20 μm apart. Arrowheads mark invasive SOX2 + cells surrounded by Laminin. Dashed line marks the approximate line of measurement of the length of the adjacent Laminin track. Sb, 25 μm and 10 μm for high-magnification image. (B) Graph showing the relative expression level of a panel of ECM- associated genes from RNA-sequencing of individual Rreb1+/+ and Rreb1-/- embryos. Each point represents a single embryo. Statistical analysis was performed using an Unpaired t-test (*p<0.05, **p<0.005, ***p<0.001). Bars represent median and IQR. Expression is shown relative to the mean expression in wild-type embryos. (E) Graph showing the top five results from KEGG pathway analysis of genes that were significantly upregulated in Rreb1-/- versus Rreb1+/+ embryos. The genes associated with each category are shown on the graph. (F) Schematic diagram summarizing some of the key findings in this paper. i. In the wild-type epiblast epithelium of the mouse embryo, adherens junction components, such as E-CADHERIN, form continuous belts along cell junctions and F-ACTIN forms linear filaments that run parallel to these junctions. ii. In Rreb1-/- embryos, there was a reduction in the expression of a cohort of factors secreted by the VE, which may alter the behavior of epiblast cells. Furthermore, we observed various phenotypes in the Rreb1-/- epiblast epithelium including a more variable cell orientation compared to that of wild-type embryos, abnormal accumulations of cells, ectopic expression of the mesenchymal marker SNAIL, and chains of cells apparently exiting the epithelial layer. iii. The wild-type epiblast epithelium forms a Laminin basement membrane at its basal surface. iv. In contrast, in chimeric embryos that contain a mix of both wild-type and Rreb1-/- cells, we observed cells of both genotypes traversing breaks in the underlying basement membrane which were then found ectopically throughout the embryo. Moreover, we observed the formation of long Laminin tracks closely associated with abnormal SOX2HI cells. v. The cell behaviors observed in Rreb1-/- embryos and chimeras are similar to those observed in cancer. For example, abnormal accumulations of epithelial cells are the basis of tumor formation, changes in cytoskeleton organization combined with a switch from linear to punctate E-CADHERIN and ectopic expression of mesenchymal markers characterizes an intermediate EMT state that is associated with collective invasion during cancer metastasis. Remodeling of the ECM into parallel fibers, known as ECM microtracks, facilitates collective cell invasion in cancer metastasis. Furthermore, the tumor microenvironment commonly show a change in the expression of secreted factors that promote angiogenesis. A, anterior; P, posterior; L, left; R, right; Pr, proximal; Ds, distal; Epi, epiblast; Endo, endoderm; ExE, extraembryonic ectoderm; Meso, mesoderm.
Figure 7—figure supplement 1. Rreb1 chimeras display changes in ECM organization.
