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Targeted deletion of beta 1 integrins in F9 embryonal carcinoma cells affects morphological differentiation but not
L E Stephens1, J E Sonne, M L Fitzgerald
1Department of Stomatology, University of California San Francisco 94143-0512.
Abstract:
The integrin superfamily of heterodimeric transmembrane adhesion receptors mediates many cell-cell and cell-matrix interactions whose functions are believed to be critical for normal morphogenesis and differentiation. By eliminating the beta 1 integrin gene through homologous recombination, we have assessed the role of the beta 1 integrin family in the F9 embryonal carcinoma model for endodermal differentiation. F9 cells were unexpectedly found to maintain three copies of the beta 1 gene and complete elimination required three sequential rounds of targeting to generate triple knockout lines (beta 1 TKO). Elimination of the beta 1 integrin family of adhesion receptors from F9 cells resulted in reduced adhesion to fibronectin, laminin and collagen, but strongly enhanced adhesion to vitronectin. The absence of beta 1 integrins did not promote significant compensatory upregulation of either beta 3 or beta 5 subunits, both of which are known to act as vitronectin receptors when associated with alpha v. The loss of beta 1 integrins severely affected morphological differentiation when the beta 1-deficient cells were induced to differentiate to either parietal or visceral endoderm. Parietal endoderm derived from beta 1-deficient cells retained a rounded morphology and migrated poorly on both fibronectin and vitronectin. Visceral endoderm derived from beta 1-deficient cells were also unable to form a normal, confluent epithelial monolayer; instead, a non-contiguous layer containing clumps of disorganized cells was observed. However, loss of beta 1 integrins did not interfere with induction by differentiating agents of tissue-specific gene products for either visceral or parietal endoderm. These results suggest that beta 1 integrins mediate morphological differentiation (migration and epithelial formation) but not tissue-specific gene expression in induced F9 cells, and that these two processes are not necessarily linked in this system.
Insights
Beta 1 integrins are crucial for cell adhesion and morphological differentiation in F9 embryonal carcinoma cells. Their absence impairs cell migration and epithelial formation but not tissue-specific gene expression during endodermal differentiation.
Area of Science:
- Cell Biology
- Developmental Biology
- Molecular Biology
Background:
- Integrins are essential transmembrane receptors mediating cell-matrix and cell-cell interactions.
- These interactions are critical for tissue morphogenesis and cellular differentiation.
- The specific role of beta 1 integrins in endodermal differentiation requires further elucidation.
Purpose of the Study:
- To investigate the function of beta 1 integrins in F9 embryonal carcinoma cells during endodermal differentiation.
- To determine the impact of beta 1 integrin loss on cell adhesion, migration, and epithelialization.
- To differentiate the roles of beta 1 integrins in morphological versus gene expression changes.
Main Methods:
- Homologous recombination was used to generate beta 1 integrin triple knockout (TKO) F9 cell lines.
- Cell adhesion assays were performed using fibronectin, laminin, collagen, and vitronectin.
- Morphological differentiation and epithelial monolayer formation were assessed after induction.
- Tissue-specific gene product induction was analyzed in beta 1-deficient cells.
Main Results:
- Complete elimination of beta 1 integrins reduced adhesion to fibronectin, laminin, and collagen, but enhanced adhesion to vitronectin.
- Beta 1 integrin deficiency severely impaired morphological differentiation, including poor migration and abnormal epithelial formation in parietal and visceral endoderm.
- Loss of beta 1 integrins did not affect the induction of tissue-specific gene products.
- No significant compensatory upregulation of beta 3 or beta 5 integrin subunits was observed.
Conclusions:
- Beta 1 integrins are essential for mediating cell adhesion and morphological differentiation processes, such as migration and epithelialization, in F9 cells.
- These integrins are not required for the induction of tissue-specific gene expression during endodermal differentiation.
- Morphological differentiation and gene expression regulation are separable processes in this model system, with beta 1 integrins playing a key role in the former.