The bilateral symmetry of the mouse embryo is broken by leftward fluid flow in the node. However, it is unclear how this directional flow is then translated into the robust, left side-specific Nodal gene expression that determines and coordinates left-right situs throughout the embryo. While manipulating Nodal and Lefty gene expression, we have observed phenomena that are indicative of the involvement of a self-enhancement and lateral-inhibition (SELI) system. We constructed a mathematical SELI model that not only simulates, but also predicts, experimental data. As predicted by the model, Nodal expression initiates even on the right side. These results indicate that directional flow represents an initial small difference between the left and right sides of the embryo, but is insufficient to determine embryonic situs. Nodal and Lefty are deployed as a SELI system required to amplify this initial bias and convert it into robust asymmetry.

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Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR)and ERBB4. Here, we show that HB-EGF-EGFR signaling is involved in eyelid development. HB-EGF expression is restricted to the tip of the leading edge of the migrating epithelium during eyelid closure in late gestation mouse embryos. Both HB-EGF null (HBdel/del) and secretion-deficient(HBuc/uc) mutant embryos exhibited delayed eyelid closure, owing to slower leading edge extension and reduced actin bundle formation in migrating epithelial cells. No changes in cell proliferation were observed in these embryos. In addition, activation of EGFR and ERK was decreased in HBdel/del eyelids. Crosses between HBdel/del mice and waved 2 mice, a hypomorphic EGFR mutant strain, indicate that HB-EGF and EGFR interact genetically in eyelid closure. Together with our data showing that embryos treated with an EGFR-specific kinase inhibitor phenocopy HBdel/del embryos, these data indicate that EGFR mediates HB-EGF-dependent eyelid closure. Finally, analysis of eyelid closure in TGFα-null mice and in HB-EGF and TGFα double null mice revealed that HB-EGF and TGFα contribute equally to and function synergistically in this process. These results indicate that soluble HB-EGF secreted from the tip of the leading edge activates the EGFR and ERK pathway, and that synergy with TGFα is required for leading edge extension in epithelial sheet migration during eyelid closure.

In mouse, left-right (L-R) patterning depends on asymmetric expression of Nodal around the node, leading to Nodal expression specifically in the left lateral plate mesoderm (LPM). Bone morphogenetic protein (BMP) signaling is also involved, but the mechanistic relationship with Nodal expression remains unclear. We find that BMP signal transduction is higher in the right LPM, although Bmp4, which is required for L-R patterning, is expressed symmetrically. By contrast, the BMP antagonists noggin (Nog) and chordin (Chrd) are expressed at higher levels in the left LPM. In Chrd;Nog double mutants, BMP signaling is elevated on both sides, whereas Nodal expression is absent. Ectopic expression of Nog in the left LPM of double mutants restores Nodal expression. Ectopic Bmp4 expression in the left LPM of wild-type embryos represses Nodal transcription, whereas ectopic Nog in the right LPM leads to inappropriate Nodal expression. These data indicate that chordin and noggin function to limit BMP signaling in the left LPM, thereby derepressing Nodal expression. In the node, they promote peripheral Nodal expression and proper node morphology, potentially in concert with Notch signaling. These results indicate that BMP antagonism is required in both the node and LPM to facilitate L-R axis establishment in the mammalian embryo.