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Kinase-negative mutant epidermal growth factor receptor (EGFR) expression during embryonal stem cell differentiation
1The Burnham Institute, La Jolla Cancer Research Center, CA 92037, USA.
Abstract:
EGF receptors are expressed on most fetal and adult cells but their precise roles are not well known. We previously reported that, in P19 embryonal carcinoma cells, the expression of kinase-negative EGFR inhibits retinoic acid (RA)-induced differentiation to nervous tissue, suggesting that EGFR plays a role in differentiation (J.-X. Wu and E. D. Adamson (1993) Dev. Biol. 159, 208-222). Embryo stem (ES) cells differentiate into a wide range of tissue types after the removal of the cytokine LIF from the culture medium. We demonstrate here that the induction of some early markers of differentiation, tissue-type plasminogen activator (tPA), AFP and keratins 8 and 19 is inhibited, whilst brachyury and myosin are increased, in clones containing kinase-negative mutant EGFR. After an extended period of differentiation, the cell types present in mutant and control cultures differed. Mutant clones produced frequent cardiac and skeletal muscle as the predominant differentiated cell types in vitro; other cells types were sparse or absent. Teratocarcinomas formed by EGFR-deltakinase-expressing ES cells contained frequent skeletal and cardiac muscle as well as apoptotic nuclei, while normal ES cells produced no detectable muscle and less apoptoses. Since mutant differentiated cultures had slower growth rates and increased levels of cell death, we concluded that: (1) inactive EGFR does not allow some cell types to survive and/or proliferate; (2) tissues that do not require EGFR for their survival, development or function predominate in long-term mutant cultures; (3) EGFR activity is not necessary for cardiac and skeletal muscle or endoderm formation and (4) Impaired survival of EGF-dependent lineages leads to preferential selection of muscle in differentiating ES cells.
Insights
Inactive epidermal growth factor receptors (EGFR) in embryonic stem cells inhibit differentiation into some cell types but promote muscle and cardiac tissue formation. This suggests EGFR is crucial for the survival of specific cell lineages during development.
Area of Science:
- Developmental Biology
- Cell Biology
- Stem Cell Research
Background:
- Epidermal growth factor receptors (EGFR) are present in fetal and adult cells, but their exact functions remain unclear.
- Previous studies indicated EGFR's role in differentiation, specifically inhibiting nervous tissue development in P19 cells when EGFR kinase activity was blocked.
Purpose of the Study:
- To investigate the role of epidermal growth factor receptor (EGFR) activity in embryonic stem (ES) cell differentiation.
- To determine how kinase-negative EGFR mutations affect the differentiation potential and cell type distribution in ES cells.
Main Methods:
- Utilized embryonic stem (ES) cell clones expressing a kinase-negative mutant epidermal growth factor receptor (EGFR).
- Assessed the expression of differentiation markers (tPA, AFP, keratins, brachyury, myosin) after LIF withdrawal.
- Analyzed cell type composition and teratocarcinoma formation in vitro and in vivo.
Main Results:
- Kinase-negative EGFR mutants showed inhibited induction of early differentiation markers (tPA, AFP, keratins) but increased brachyury and myosin.
- Long-term differentiation cultures of mutant ES cells predominantly formed cardiac and skeletal muscle.
- Teratocarcinomas derived from mutant ES cells contained more muscle tissue and apoptotic nuclei compared to controls.
- Mutant cultures exhibited slower growth rates and increased cell death.
Conclusions:
- Inactive EGFR impairs the survival and proliferation of certain cell types.
- Tissues not dependent on EGFR signaling for development become predominant in long-term cultures with inactive EGFR.
- EGFR activity is not essential for the formation of cardiac muscle, skeletal muscle, or endoderm.
- Impaired survival of EGF-dependent cell lineages results in the preferential selection of muscle cells during ES cell differentiation.