Fig. 4. S1P_1-3 are expressed in the dorsal pancreatic mesenchyme. (A) RT-PCR detection of mRNAs for S1P_1-5 in 10.5 dpc wild-type dorsal pancreatic mesenchyme. Mesenchyme of the dorsal pancreas was separated from the endoderm and total RNA was isolated. Analyses of Isl1 and ß-actin mRNAs were used as positive controls, whereas detection of Pdx1 mRNA was used for excluding endoderm contamination. The presence or absence of reverse transcriptase is indicated by (+) and (-), respectively. (B-J) Double in situ hybridization and immunofluorescence analysis of S1P₁ (B), S1P₂ (E) and S1P₃ (H) mRNA and endomucin (marker for endothelial and hematopoietic progenitor cells; C,F,I) (Brachtendorf et al., 2001), respectively, in 10.5 dpc wild-type embryos. (D,G,J) Overlay of in situ hybridization and endomucin staining images to visualize possible colocalization of S1P₁ (D), S1P₂ (G) and S1P₃ (J) with endothelial cells. Whereas S1P₁ mRNA was localized in the endothelium of major and minor blood vessels in the embryo, such as the aorta (not shown) and vessels in the mesenchyme surrounding the dorsal pancreas (B-D), mRNAs for S1P₂ and S1P₃ were expressed in the mesenchyme surrounding the dorsal pancreas (E-J). Arrowheads indicate colocalization of S1P₁ mRNA with endothelial cells in D, and absence of S1P₂ and S1P₃ in endothelial cells in G and J, respectively. The dorsal pancreatic endoderm is indicated by striped lines. Scale bar in H: 10 µm for B-J.