Between the time of implantation and gastrulation, the pluripotent cells of the mammalian epiblast become restricted to specific lineages in a series of inductive interactions that depend on both intercellular signals and highly orchestrated cell rearrangements. One day after implantation (e5.5), the embryonic region that will give rise to the three germ layers of the mouse is a single-layered cup-shaped columnar epithelium (the epiblast) that is surrounded by the squamous visceral endoderm (VE) epithelium. At this stage, the mouse embryo is elongated in its proximal-distal axis, where the site of connection to the uterine tissue defines the proximal pole. Proximal-distal differences in the pattern of gene expression first become apparent at e5.5, when a group of VE cells at the distal tip of the embryo (the distal visceral endoderm (DVE)) expresses a distinctive set of molecular markers, including the transcription factor Hex. Between e5.5 and e6.0, this population of cells migrates proximally and comes to lie on the presumptive anterior side of the embryo, adjacent to the embryonic/extra-embryonic boundary 
, where the cells are known as the anterior visceral endoderm (AVE). The cells of the AVE secrete localized Nodal and Wnt inhibitors that confine Wnt and Nodal signals to the opposite side of the embryo, where the primitive streak is then specified. Thus migration of DVE/AVE cells converts the early proximal-distal asymmetry into the definitive anterior-posterior (AP) axis of the animal.
Although migration of mammalian cells has been studied extensively in culture, little is known about the dynamics of cell migration in intact mouse embryos. As the AVE lies on the surface of the embryo and AVE migration is completed in about 5 h, it has been possible to image migration of AVE cells in vivo 
. The AVE is therefore ideal for studies of the cell behaviors and the genetic control of mammalian cell migration in vivo.
The mechanisms of AVE migration are the subject of considerable debate. It has been observed that migrating AVE cells extend filopodia in the direction of movement, which suggests that they may migrate towards an unidentified chemoattractant 
or away from a chemorepellant 
. Alternatively, it has been proposed that the cells might migrate in response to instructive cues from the extracellular matrix 
. The non-canonical Wnt pathway proteins Celsr1 and Testin are expressed in the AVE 
, which suggests that AVE cells might move through planar polarity-dependent cellular rearrangements analogous to those that take place in the extending germ band of Drosophila 
. In mouse embryos that lack Prickle1, a non-canonical Wnt pathway protein, the AVE fails to move; however, this defect is accompanied by a disruption of apical-basal polarity, so it is not clear whether Prickle1 regulates AVE migration directly 
We showed previously that the actin regulator Nap1, a component of the WAVE complex, is important for AVE migration 
. The WAVE complex is crucial for migration of many cell types; it promotes the formation of branched actin networks at the leading edge of migrating cells and thereby pushes the cell membrane forward 
. The ENU-induced Nap1khlo
allele was identified in a genetic screen for mutations that affect embryonic patterning 
. The most dramatic phenotype of Nap1khlo
mutants is the duplication of the AP body axis, seen in about 25% of the mutant embryos, and correlated with partial migration of the AVE. These studies indicated that WAVE-mediated migration was important for AVE migration and axis specification, but the low penetrance of the axis duplication phenotype left open the possibility that other, WAVE-independent mechanisms might be crucial for AVE migration. The WAVE complex can act downstream of Nck or Rac 
; activation by Rac requires simultaneous interaction with acidic phospholipids 
. Because the AP body axis is specified normally in mouse mutants that lack Nck 
, we hypothesized that the small GTPase Rac1 might act upstream of the WAVE complex to direct AVE migration.
The functions of mammalian Rac1 have been studied in a variety of processes. Conditional inactivation of Rac1
in mouse fibroblasts has confirmed that Rac plays roles in lamellipodia formation, cell-matrix adhesion, and cell survival 
. Tissue-specific gene inactivation experiments have also implicated mouse Rac1 in a variety of processes, including EGF-induced cell proliferation of the neural crest 
, canonical Wnt signaling during limb outgrowth 
, actin rearrangements required for myoblast fusion 
, regulation of p38 MAP kinase activity and of Indian Hedgehog expression in chondrocytes 
, and maintenance of stem cells in the skin through regulation of c-Myc expression 
. Nevertheless, no genetic studies have addressed the roles of Rac proteins in vertebrate morphogenesis.
Although the mouse genome encodes three forms of Rac, only Rac1
is expressed in the early embryo 
. Because the tissue organization of the early mouse embryo is relatively simple, we set out to define precise functions of Rac in tissue migration in vivo in the intact early embryo. It was previously shown that Rac1
null embryos die at the time of gastrulation 
. Here we show that Rac1
is essential for the establishment of the AP axis of the mouse embryo. We use high-resolution imaging to show that wild-type AVE cells move in a coordinated fashion and extend long projections that can span nearly the entire embryonic region. In embryos that lack Rac1
, AVE cells lack all projections and fail to move from their original location at the distal tip of the embryo. These findings demonstrate that Rac1 is a critical link that connects morphogenetic signals to AVE migration, allowing the establishment of the AP axis of the mammalian body plan.