Expression of functional c-kit receptors rescues the genetic defect of W mutant mast cells
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W S Alexander1, S D Lyman, E F Wagner
1Research Institute for Molecular Pathology (IMP), Vienna, Austria.
Abstract:
Loss-of-function mutations in the gene for the c-kit tyrosine kinase receptor are strongly implicated in the developmental abnormalities of W mutant mice. To dissect further the relationship between kit and the W phenotype, retroviruses carrying the normal murine c-kit gene were constructed. In infected cells, the level of c-kit expression from these vectors varied markedly with different promoter elements, the 5' viral LTR proving to be the most effective. When introduced into cells which normally do not express c-kit, ectopic kit receptors transduced a ligand (Steel factor)-dependent proliferative signal in IL-3-dependent DA-1 myeloid cells and induced transformation in fibroblasts. Primary mutant mast cells were used to examine the effects of reconstituting functional kit expression in cells affected by W mutations. Exogenous c-kit expression rescued the defective proliferative response to Steel factor of cells from both W/Wv and W/W mutant mice. Moreover, functional kit expression also restored the capacity of W/Wv mast cells to survive and differentiate in vivo. These results imply that defective c-kit receptor function is sufficient to generate the W mutant phenotype.
Insights
Defective c-kit receptor function causes developmental abnormalities in W mutant mice. Restoring functional c-kit expression rescued cellular defects and restored normal development in these mice.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- Loss-of-function mutations in the c-kit tyrosine kinase receptor gene are linked to W mutant mouse developmental abnormalities.
- Understanding the precise role of c-kit in these phenotypes requires further investigation.
Purpose of the Study:
- To investigate the relationship between c-kit gene function and the W phenotype.
- To determine if restoring functional c-kit expression can rescue cellular defects associated with W mutations.
Main Methods:
- Construction of retroviruses carrying the normal murine c-kit gene.
- Transduction of various cell types, including IL-3-dependent DA-1 myeloid cells, fibroblasts, and primary mutant mast cells.
- Analysis of c-kit expression levels and functional rescue of Steel factor-dependent signaling and cellular differentiation.
Main Results:
- The 5' viral LTR was identified as the most effective promoter for c-kit expression from retroviral vectors.
- Ectopic c-kit expression in cells lacking normal expression induced Steel factor-dependent proliferation and fibroblast transformation.
- Exogenous c-kit expression rescued the defective Steel factor response in mast cells from W/Wv and W/W mutant mice.
- Functional c-kit expression restored in vivo survival and differentiation of W/Wv mast cells.
Conclusions:
- Defective c-kit receptor function is sufficient to cause the W mutant phenotype.
- Restoration of functional c-kit signaling can rescue cellular defects and restore normal development in W mutant mice.