The elaboration of the major axes of the embryo (anterior-posterior, dorsal-ventral, and left-right) occurs at the time of gastrulation, the morphogenetic process that forms the primary germ layers (ectoderm, mesoderm, and definitive endoderm). The molecular definition of a left-right (LR) axis precedes the establishment of an overt asymmetry in that dimension, a common feature across bilateral animals. Despite some species-specific differences, the sequence of key events and core components involved in the establishment of LR asymmetry appears conserved across vertebrates 
An initial LR symmetry-breaking event occurs in a specialized organ located at the embryonic midline, this being the node in the mouse. Around embryonic day (E) 7.75, equivalent to the early head-fold (EHF) stage, cilia protruding from the posterior-apical surface of node cells begin rotating, thereby generating directional fluid flow within the node microenvironment 
. Whether through interpretation of mechanical flow forces 
or asymmetric distribution of signals 
, nodal flow is believed to trigger a wave of Ca2+
on the left side of the node 
, as well as left-biased asymmetric perinodal expression of Nodal
. By E8.5, equivalent to the ~4 somite stage, an asymmetric readout of this symmetry-breaking event is the activation of the Nodal/Lefty2/Pitx2
genetic cascade within the left lateral plate mesoderm (LPM) 
. Activation of these factors is required for asymmetric organogenesis 
. The embryonic midline is thought to act as a “midline barrier” in keeping signals confined to their respective sides, both by virtue of its morphological structure and by expressing specific factors 
An unresolved question in the establishment of LR asymmetry in the mouse embryo is the mechanism of communication between the midline site of symmetry-breaking and the lateral plate tissues initiating asymmetric morphogenesis. By virtue of their location, cells that lie between the node and lateral plate are likely to provide the medium for signal relay. The gut endoderm and paraxial mesoderm are attractive candidate tissues for mediating signal transmission since they lie adjacent to both the node and lateral plate mesoderm. Moreover, perinodal asymmetric events, including calcium release and Nodal
expression, occur in endodermal cells lining the node 
Using live imaging and genetic labeling, we previously noted that the gut endoderm of the mouse embryo forms by widespread intercalation of epiblast-derived definitive endoderm (DE) cells into the overlying visceral endoderm (VE) epithelium 
. At gastrulation, DE progenitors intercalate into the distally positioned embryonic VE epithelium, also referred to as the emVE 
, which constitutes the surface cell layer of the embryo. This multifocal intercalation of DE cells leads to the widespread dispersal and dilution of emVE cells 
. As a result, the emergent gut endoderm tissue is comprised of cells of two distinct origins: DE and emVE. Furthermore, we noted that in addition to their scattered distribution within the gut endoderm, residual emVE cells exhibited a second stereotypic distribution in that they were absent from, but congregate around, the node and midline 
. Even though the precise cellular dynamics orchestrating emVE displacement at the node and midline remain unknown, these data suggest that lateral dispersal in the future gut endoderm and midline displacement in the future notochord may be regulated independently.
-related HMG box transcription factor Sox17 is a key conserved factor involved in endoderm formation in vertebrates 
. Mice lacking Sox17
have a depletion of DE cells, possess an abnormal gut tube, and die around E10.5 
. Interestingly, Sox17
mutant mouse embryos also display gross morphological features commonly observed in mutants of LR asymmetry establishment, including a failure to turn from a lordotic to a fetal position, an open body wall, and cardiac defects 
. We therefore reasoned that a detailed analysis of the Sox17
mutant might provide further insight into the gut endoderm defects and whether gut endoderm morphogenesis and LR patterning are coupled.
Here we report that Sox17 mutant embryos exhibit a failure in emVE dispersal as well as defects in LR patterning. We noted that widespread emVE and DE cell intercalation in the prospective gut endoderm was severely affected in mutants. By contrast, emVE displacement at the midline was not. This suggested that lateral dispersal in the future gut endoderm and midline displacement in the future notochord are likely to be distinct morphogenetic processes, the former of which requires Sox17.
One mode of communication across epithelia is through gap junctions. We identified Connexin43 (Cx43) as the predominant gap junctional constituent expressed in the endoderm at a time soon after widespread emVE and DE cell intercalation is complete, when node to LPM signal relay likely occurs. In Sox17 mutants, we noted that Cx43 is absent in the gut endoderm. We demonstrated gap junctional coupling on both the left and right sides of the gut endoderm of wild-type embryos, but not in Sox17 mutants. We also observed that gap junction coupling in the mesoderm was isolated from the endoderm. Since Cx43 localization within the mesoderm was comparable in wild-type and mutant embryos, we concluded that LR signals must be propagated across the endoderm epithelium. Our studies also revealed an absence of gap junctional coupling across cells at the midline in wild-type embryos, thereby providing the first functional visualization of a midline barrier in the mouse.
Collectively our observations identify the gut endoderm as a key tissue of communication between node and LPM during the establishment of LR asymmetry in the mouse. We demonstrate that Cx43-mediated gap junction coupling across the endoderm is necessary for the correct temporal and spatial propagation of asymmetric signal(s) from the node to the LPM.