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First published online 2 February 2005
doi: 10.1242/dev.01643

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Vascular function and sphingosine-1-phosphate regulate development of the dorsal pancreatic mesenchyme

Josefina Edsbagge^1,*, Jenny K. Johansson^1,*,, Farzad Esni^1,, Yang Luo², Glenn L. Radice² and Henrik Semb^1,,

¹ Department of Medical Biochemistry, Box 440, Göteborg University, S-405 30 Göteborg, Sweden
² Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, 1355 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, PA 19104, USA

View larger version (78K): [in a new window]   Fig. 1. Restored cardiac/vascular function in N-cadherin-deficient embryos rescues formation of the dorsal pancreas. Histochemically and immunohistochemically stained sections of the dorsal pancreatic region from 9.5 dpc wild-type (A,D,G,J,M), Cdh2-/- (B,E,H,K,N), cardiac-rescued Cdh2-/- (Cdh2-/-Tg) (C,F,I,L,O) embryos, and from 10.5 dpc wild type (P) and cardiac-rescued Cdh2-/- (Q) embryos. (A-C) Sections were stained with hematoxylin and eosin (HE). (D-Q) Immunohistochemical staining of sections with antibodies against Isl1 (D-F,P,Q), Pdx1 (G-I), N-cadherin (Cdh2; J-L; inset shows expression of the Cdh2 transgene in the heart) and CD31 (M-O). Despite the lack of N-cadherin expression within the dorsal pancreatic mesenchyme, dorsal pancreas formation is rescued in cardiac-rescued Cdh2-/- embryos (G-L). The dorsal pancreatic endoderm and dorsal aorta are indicated by broken line and asterisk, respectively. Scale bars: in A, 30 µm for A-C; in D, 20 µm for D-O; in P, 20 µm for P,Q; inset in L, 40 µm.

View larger version (28K): [in a new window]   Fig. 2. 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), 0.2 µg/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: 100 µm.

View larger version (46K): [in a new window]   Fig. 3. 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.

View larger version (118K): [in a new window]   Fig. 4. S1P1-3 are expressed in the dorsal pancreatic mesenchyme. (A) RT-PCR detection of mRNAs for S1P1-5 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.

View larger version (29K): [in a new window]   Fig. 5. 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, 20 µm for B,C; in D, 20 µm for D,E.

View larger version (48K): [in a new window]   Fig. 6. 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.

View larger version (44K): [in a new window]   Fig. 7. Proposed model for how blood vessel function contributes to pancreas development subsequent to endoderm specification. Blood vessel (aorta)-derived S1P binds S1P receptors on dorsal pancreatic mesenchymal cells (1) or endothelial cells (2), resulting in heterotrimeric G-protein-mediated intracellular signalling and enhanced mesenchymal cell proliferation/survival in a cell-autonomous manner (1) or indirectly via S1PR-triggered signalling from endothelial cells (2). Mesenchymal cells accumulate around the committed dorsal pancreatic endoderm and secrete soluble factors necessary for further growth/differentiation of the endoderm. Da, dorsal aorta; m, mesenchymal cells; e, endothelial cells; dp, dorsal pancreas; arrows, recruitment of dorsal pancreatic mesenchymal cells from lateral plate mesenchyme.