Plasma and S1P rescue early morphogenesis of the dorsal pancreas in Cdh2^-/- explants. Pancreatic-gut explants from 9.5 dpc wild-type (WT; A,G) and Cdh2^-/- (B-F) embryos stained with Pdx1 antibodies. (B-F) Explants from 9.5 dpc Cdh2^-/- embryos incubated in the presence of beads soaked in PBS (negative control; C), plasma from 15.5 dpc wild-type embryos (D), 0.1 μM S1P (E), 0.2μ g/ml PTX+0.1 μM S1P (PTX beads were pre-grafted 2 hours before S1P beads were added) (F), and explants from 9.5 dpc wild-type embryos incubated with (G) or without (A) beads soaked in 0.2 μg/ml PTX. The beads were grafted on the dorsal side of the gut, at the site of the presumptive dorsal pancreas. After 48 hours in culture, the explants were stained with Pdx1 and photographed. Vb, ventral pancreatic bud; db, dorsal pancreatic bud; s, stomach; i, intestine; gb, gall bladder; pla, plasma. Scale bar in A: 100μ m.

Plasma and S1P cannot induce growth/differentiation of mesenchyme-free dorsal pancreatic endoderm. Isolated intact (A) or mesenchyme-stripped wild-type 10.5 dpc dorsal pancreatic bud were cultured for 48 hours with isolated 10.5 dorsal pancreatic mesenchyme (B), no addition (C), with beads soaked in PBS (D), plasma (E), or 0.1 μM S1P (F) and stained with Pdx1 antibodies. Whereas the intact dorsal pancreatic bud (A) as well as mesenchyme-free dorsal pancreatic bud endoderm recombined with dorsal pancreatic mesenchyme (B) grew, mesenchyme-free dorsal pancreatic endoderm failed to grow alone (C) or in the presence of PBS (D), plasma (E) and S1P (F), respectively. Of note, in B the mesenchyme was lost during the staining procedure. e and end, dorsal pancreatic endoderm; m and mes, dorsal pancreatic mesenchyme; pla, plasma from 15.5 wild-type embryos. Scale bar: 50 μm.

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 andβ -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.

S1P stimulates proliferation of dorsal pancreatic mesenchymal cells in a pertussis toxin-sensitive manner. (A-C) Primary cultures of 10.5 dpc wild-type dorsal pancreatic mesenchymal cells were established and incubated with or without 0.5 μM S1P or 0.2 μg/ml PTX (including a 2 hour pre-incubation)+0.5 μM S1P for 24 hours. Quantification of the total number of cells and the number of proliferating cells were estimated by counting DAPI- and BrdU-positive cells, respectively. Data are presented as fold increase over control. Asterisks indicate that S1P significantly increases cell proliferation and that PTX inhibits this effect, as determined by two-tailed t-test (P<0.05). Error bars represent s.e.m. To detect proliferating mesenchymal and endothelial cells, double immunofluorescence was performed with Isl1 (mesenchymal marker) and Ki67 (B), and CD31 (endothelial marker) and Ki67 (C). The ratio of mesenchymal and endothelial cells was estimated to be 9:1. Although both mesenchymal and endothelial cells underwent mitosis, the majority of the proliferating cells were of mesenchymal cell origin. (D,E) To detect changes in the migratory behaviour of the mesenchymal cells, F-actin was visualised by Phalloidin staining after a 24-hour incubation with (E) or without (D) 0.5 μM S1P. No change in F-actin rearrangement could be distinguished. Scale bars: in B, 20μ m for B,C; in D, 20 μm for D,E.

Blood vessels are present but disorganized around Cdh2^-/- pancreatic buds independent of the presence or absence of S1P. Pancreatic-gut explants from wild type (WT; A) and Cdh2^-/- embryos incubated with beads soaked in PBS (B) or S1P (C) were analysed for the presence and distribution of blood vessels by double immunostainings with CD31 (blue) and Pdx1 (brown) antibodies. Higher magnification of the pancreatic region in A, B, and C is shown in D, E, and F, respectively. Whereas blood vessels were present nearby the pancreatic endoderm independent of the genotype and presence of S1P, N-cadherin-deficiency resulted in a disorganised network of blood vessels. S1P stimulated an overall growth of the pancreatic-gut explants, including endoderm, mesenchyme and blood vessels. db, dorsal pancreatic bud; vb, ventral pancreatic bud; gb, gall bladder. Both the dorsal (left) and ventral (right) pancreas is delineated with red broken lines. Scale bars: in A, 100 μm for A-C; in D, 30 μm for D-F.