Roles for Rho/ROCK and vinculin in parietal endoderm migration.
The first cell migration event in the mouse embryo is the movement of parietal endoderm cells from the surface of the inner cell mass facing the blastocoel cavity to line the inner surface of the trophectoderm. F9 embryoid bodies provide an in vitro model for this event. They have an inner core of undifferentiated stem cells surrounded by an outer visceral endoderm layer. When plated on a laminin coated substrate, visceral endoderm transitions to parietal endoderm and migrates onto the dish, away from the attached embryoid body. We now show that this outgrowth contains abundant focal complexes and focal adhesions, as well as lamellipodia and filopodia. Treatment with the ROCK inhibitor Y-27632 promotes a 2-fold increase in outgrowth, and a transition from focal adhesions and associated stress fibers, to focal complexes and a decrease in stress fibers. ROCK inhibition also leads to an increase in lamellipodia. Inhibition of RhoA by transfection of a vector encoding C3 transferase, direct administration of the C3 enzyme, or transfection of a vector encoding p190 Rho GTPase Activating Protein also promotes outgrowth and an apparent transition from focal adhesions to focal complexes. Parietal endoderm outgrowth generated using vinculin-deficient F9 stem cells migrates 2-fold further than wild type cultures, but this outgrowth retains the morphology of wild type parietal endoderm, including focal adhesions and stress fibers. Addition of Y-27632 to vinculin-null outgrowth cultures further stimulates migration an additional 2-fold, supporting the conclusion that Rho/ROCK and vinculin regulate parietal endoderm outgrowth by distinct pathways.
Mills
Evan
E
Department of Biology, Wesleyan University, Middletown, Connecticut 06459-0170, USA.
LaMonica
Kristi
K
Hong
Tao
T
Pagliaruli
Thomas
T
Mulrooney
James
J
Grabel
Laura
L
D036002:ADP Ribose Transferases / Q000378:metabolism; D000818:Animals; D001905:Botulinum Toxins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002448:Cell Adhesion; D002465:Cell Movement; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D004707:Endoderm / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011554:Pseudopodia / Q000378:metabolism; D012097:Repressor Proteins; D018243:Teratocarcinoma / Q000473:pathology; D016596:Vinculin / Q000172:deficiency / Q000378:metabolism*; D054460:rho-Associated Kinases
C513041:Arhgap35 protein, mouse; C513042:Arhgap5 protein, mouse; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D047908:Intracellular Signaling Peptides and Proteins; D012097:Repressor Proteins; D016596:Vinculin; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D001905:Botulinum Toxins