Fig. 4. Immunofluorescence detection of 3xMyc-tagged Nodal at the node. (A) Schematic representation of a Nodal transgene. Tandem node-specific enhancers (NDEs) were linked with the hsp68 promoter, Nodal cDNA (encoding the 3xMyc tag positioned four amino acids downstream from the Nodal cleavage site, arrowhead), IRES, lacZ and pA. (B) An E8.2 mouse embryo harboring the transgene (Tg⁺) exhibits ß-galactosidase activity only at the node. (C-E) In situ hybridization for Nodal (C,D) or Pitx2 (E) mRNA in Nodal^neo/neo (C) or Nodal^neo/neo; Tg⁺ (D,E) embryos at E8.2. The expression of the transgene (black arrowhead) rescues the loss of Nodal and Pitx2 expression in LPM (red arrowheads). The level of expression of the rescued Nodal and Pitx2 is lower than that in the wild-type embryo because the neo gene inserted into the endogenous Nodal gene prevents Nodal expression in the LPM from being amplified to the maximum level. (F-J) Transverse frozen sections of E8.0 Tg⁺ embryos were subjected to immunofluorescence analysis either with antibodies to Myc (F) and to ß-galactosidase (G), with the merged image shown in H, or with antibodies to Myc and to ZO-1 (I) or laminin (J), with the fluorescence signals being merged with the differential interference contrast and DAPI image (blue) in I,J. The basement membrane is indicated by white dots (I). c, crown cell of the node; ec, ectoderm; m, mesoderm. Scale bars: 200 µm in B-E; 20 µm in F-H; 5 µm in I-J.