| Abstrakt: |
The establishment of the parietal endoderm (PE) layer within the rodent blastocyst requires extensive outward migration of inner cell mass (ICM)-derived primitive endoderm (PrE) cells over the blastocoelic surface of the trophectoderm. While a role for Reichert’s membrane in this process has been proposed, the identity of the basement membrane component(s) involved and the mechanism(s) by which they might promote and/or guide this movement remain to be determined. This study was a comparison of the gradient-associated influences of three components of Reichert’s membrane—fibronectin, laminin, and collagen IV—on the migration of embryonic endoderm-like cells obtained from the F9 embryocarcinoma cell line. Gradients (positive or negative) or even coatings of each of these glycoproteins were established across polycarbonate filters; the filters were then placed in blind-well chambers. F9 cells, either undifferentiated or pretreated with retinoic acid (RA) alone or RA+dibutyryl cAMP to resemble PrE and PE, respectively, were then loaded into the upper chamber compartments. At the end of the migration assay ( 4–24 h), the number of cells that had moved through the filter pores and attached to the lower filter surface was determined for each treatment group.While even coatings of either fibronectin or laminin supported some cell migration through the filters, these levels were increased 13- and 20-fold (fibronectin and laminin, respectively) by application of the glycoproteins as positive gradients. In contrast, no migration occurred in response to negative gradients of either fibronectin or laminin. The extent of cell migration increased in response to duration of the assay and the level of cell differentiation. Minimal coating concentrations of 0.2 μg/ml (fibronectin) and 0.4 μg/ml (laminin) were required to support both cell attachment and gradient-directed translocation; maximal migratory responses were obtained with I and 2 μg/ml coating concentrations, respectively, of these glycoproteins. Unlike fibronectin and laminin, collagen IV provided minimal support of cell attachment and no gradient-directed stimulation of translocation, irrespective of the differentiated status of the cells. These findings indicate that the in vitro migration of F9-derived PrE- and PE-like cells is promoted by positive gradients of fibronectin and laminin, two components of Reichert’s membrane. Within the blastocyst, the establishment of similar gradients from embryonic to abembryonic poles may provide the means of promoting and guiding the outward migration of ICM-derived PrE cells to establish the PE layer. |
