The Wsh and Ph mutations affect the c-kit expression profile: c-kit misexpression in embryogenesis impairs...

Area of Science:

• Genetics
• Developmental Biology
• Molecular Biology

Background:

• Receptor tyrosine kinases (RTKs) c-kit and platelet-derived growth factor receptor alpha chain (PDG-FRa) are encoded at the white spotting (W) and patch (Ph) loci on mouse chromosome 5.
• W mutations impact melanogenesis, gametogenesis, and hematopoiesis, while Ph mutations affect melanogenesis and cause early lethality in homozygotes.
• The W-sash (Wsh) mutation alters c-kit expression, blocking it in some cells and enhancing it in others crucial for melanogenesis.

Purpose of the Study:

• To determine the effect of the Ph mutation on c-kit expression during embryogenesis in heterozygotes.
• To investigate the molecular basis of the Wsh mutation and the organization of RTKs in the W/Ph region.
• To elucidate the role of c-kit misexpression in pigment deficiency observed in Wsh and Ph mutant mice.

Main Methods:

• Immunohistochemical analysis to assess c-kit expression in Ph heterozygotes.
• Pulsed-field gel electrophoresis to characterize the Wsh mutation and gene organization.
• Genetic mapping to define the deletion endpoints of the Ph mutation.

Main Results:

• Immunohistochemistry revealed enhanced c-kit expression in key melanogenesis sites in Ph heterozygotes.
• The RTK gene order was determined as Pdgfra-c-kit-flk1.
• Wsh mutation results in unlinked c-kit and Pdgfra genes, suggesting an inversion.
• The Ph mutation involves a deletion including Pdgfra, with its endpoint between Pdgfra and c-kit.

Conclusions:

• Misexpression of c-kit, driven by both Wsh and Ph mutations, is proposed to cause pigment deficiency by affecting early melanogenesis.
• The Wsh mutation affects positive regulatory elements of c-kit, while both Wsh and Ph mutations impact negative regulatory elements.
• Genetic analysis defined the chromosomal organization and characterized specific mutations affecting RTK function and expression.