lefty-1, lefty-2, and nodal are expressed on the left side of developing mouse embryos and are implicated in left-right (L-R) determination. The role of lefty-1 was examined by analyzing mutant mice lacking this gene. The lefty-1-deficient mice showed a variety of L-R positional defects in visceral organs. Unexpectedly, however, the most common feature of lefty-1-/- mice was thoracic left isomerism (rather than right isomerism). The lack of lefty-1 resulted in bilateral expression of nodal, lefty-2, and Pitx2 (a homeobox gene normally expressed on the left side). These observations suggest that the role of lefty-1 is to restrict the expression of lefty-2 and nodal to the left side, and that lefty-2 or nodal encodes a signal for "leftness."

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.

The left-right (L-R) asymmetric expression of lefty2 and nodal is controlled by a left side-specific enhancer (ASE). The transcription factor FAST2, which can mediate signaling by TGF beta and activin, has now been identified as a protein that binds to a conserved sequence in ASE. These FAST2 binding sites were both essential and sufficient for L-R asymmetric gene expression. The Fast2 gene is bilaterally expressed when nodal and lefty2 are expressed on the left side. TGF beta and activin can activate the ASE activity in a FAST2-dependent manner, while Nodal can do so in the presence of an EGF-CFC protein. These results suggest that the asymmetric expression of lefty2 and nodal is induced by a left side-specific TGF beta-related factor, which is most likely Nodal itself.

FoxH1 (FAST) is a transcription factor that mediates

Mouse anterior-posterior axis polarization is preceded by formation of the distal visceral endoderm (DVE) by unknown mechanisms. Here, we show by in vitro culturing of embryos immediately after implantation in microfabricated cavities that the external mechanical cues exerted on the embryo are crucial for DVE formation, as well as the elongated egg cylinder shape, without affecting embryo-intrinsic transcriptional programs except those involving DVE-specific genes. This implies that these developmental events immediately after implantation are not simply embryo-autonomous processes but require extrinsic factors from maternal tissues. Moreover, the mechanical forces induce a breach of the basement membrane barrier at the distal portion locally, and thereby the transmigrated epiblast cells emerge as the DVE cells. Thus, we propose that external mechanical forces exerted by the interaction between embryo and maternal uterine tissues directly control the location of DVE formation at the distal tip and consequently establish the mammalian primary body axis.