Increased basal cAMP-dependent protein kinase activity inhibits the formation of mesoderm-derived structures in the developing mouse embryo.
A targeted disruption of the RIalpha isoform of protein kinase A (PKA) was created by using homologous recombination in embryonic stem cells. Unlike the other regulatory and catalytic subunits of PKA, RIalpha is the only isoform that is essential for early embryonic development. RIalpha homozygous mutant embryos fail to develop a functional heart tube at E8.5 and are resorbed at approximately E10.5. Mutant embryos show significant growth retardation and developmental delay compared with wild type littermates from E7.5 to E10.5. The anterior-posterior axis of RIalpha mutants is well developed, with a prominent head structure but a reduced trunk. PKA activity measurements reveal an increased basal PKA activity in these embryos. Brachyury mRNA expression in the primitive streak of RIalpha mutants is significantly reduced, consistent with later deficits in axial, paraxial, and lateral plate mesodermal derivatives. This defect in the production and migration of mesoderm can be completely rescued by crossing RIalpha mutants to mice carrying a targeted disruption in the Calpha catalytic subunit, demonstrating that unregulated PKA activity rather than a specific loss of RIalpha is responsible for the phenotype. Primary embryonic fibroblasts from RIalpha mutant embryos display an abnormal cytoskeleton and an altered ability to migrate in cell culture. Our results demonstrate that unregulated PKA activity negatively affects growth factor-mediated mesoderm formation during early mouse development.
Amieux
Paul S
PS
Department of Pharmacology, University of Washington, Seattle, WA 98195, USA.
Howe
Douglas G
DG
Knickerbocker
Heidi
H
Lee
David C
DC
Su
Thomas
T
Laszlo
George S
GS
Idzerda
Rejean L
RL
McKnight
G Stanley
GS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D020134:Catalytic Domain; D002453:Cell Cycle; D002465:Cell Movement; D002851:Chromatography, High Pressure Liquid; D003433:Crosses, Genetic; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology*; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D008648:Mesoderm / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020033:Protein Isoforms; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D020033:Protein Isoforms; D017868:Cyclic AMP-Dependent Protein Kinases