Rho kinase activity controls directional cell movements during primitive streak formation in the rabbit embryo.

During animal gastrulation, the specification of the embryonic axes is accompanied by epithelio-mesenchymal transition (EMT), the first major change in cell shape after fertilization. EMT takes place in disparate topographical arrangements, such as the circular blastopore of amphibians, the straight primitive streak of birds and mammals or in intermediate gastrulation forms of other amniotes such as reptiles. Planar cell movements are prime candidates to arrange specific modes of gastrulation but there is no consensus view on their role in different vertebrate classes. Here, we test the impact of interfering with Rho kinase-mediated cell movements on gastrulation topography in blastocysts of the rabbit, which has a flat embryonic disc typical for most mammals. Time-lapse video microscopy, electron microscopy, gene expression and morphometric analyses of the effect of inhibiting ROCK activity showed - besides normal specification of the organizer region - a dose-dependent disruption of primitive streak formation; this disruption resulted in circular, arc-shaped or intermediate forms, reminiscent of those found in amphibians, fishes and reptiles. Our results reveal a crucial role of ROCK-controlled directional cell movements during rabbit primitive streak formation and highlight the possibility that temporal and spatial modulation of cell movements were instrumental for the evolution of gastrulation forms.

Invasion by matrix metalloproteinase-expressing cells is important for primitive streak formation in early chick blastoderm.

Epiblast cells in the early chick embryo differentiate to form all three germ layers through ingression of cells at the primitive streak across the basement membrane that underlies the epiblast. We tested the idea that degradation of the extracellular matrix components by matrix metalloproteinase(s) (MMPs) is involved in this process. Epiblast cells and primitive streak cells were dissociated into single cells and seeded onto a reconstituted basement membrane gel in vitro. Following overnight culture, approximately half the cells made holes in the substratum by dissolving the gel matrix. This invasive phenomenon was reproduced in vitro even when the cells were cultured upside down using a hanging culture system. We detected gelatinase activity in the culture supernatants from both prestreak epiblast cells and primitive streak cells. Pro-MMP-2 was detected in the culture media of the prestreak/streak cells as a 72-kDa band by gelatin zymography. In RT-PCR experiments, mRNAs for MMP-2, membrane-type (MT)3-MMP and MMP-11(stromelysin-3) were expressed in the epiblast cells before and during primitive streak formation. Injection of GM 6001 or other MMP inhibitors into the subgerminal cavity of the embryo inhibited the formation of the primitive streak and/or the primitive groove in more than 82% of the injected embryos. On the other hand, injection of a negative control compound instead of GM 6001 did not cause substantial inhibition. These results suggest that MMPs are involved in the enzymatic degradation of the basement membrane underlying the epiblast and are thus important for the ingression of mesendodermal cells along the primitive streak.