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Figures
- Fig. 1.
Dorsal Pdx1-positive endoderm cells interact with endothelial cells more extensively than ventral Pdx1-positive endoderm cells. Double cell labeling for Pdx1 (DAB immunostaining, brown) and β-galactosidase (X-gal, blue) on transverse sections of Flk1lacZ/+ embryos at E8.5-9.5 (somite stages are indicated on the top of each panel). nc, notochord. The data shown are representative of multiple embryos assayed and sections throughout midgut regions. The boxed regions in D,F,H,J,L,N are magnified in E,G,I,K,M,O, respectively. (A, F) Before the emergence of Pdx1-positive cells, the aorta only has limited contact with the dorsal endoderm, laterally. (B,C) By 12-15S, the aorta moves medially and interacts extensively with the endoderm in a portion of the Pdx1-positive domain, first evident at 15S. No mesenchyme cells were detected between portions of the endoderm and the aorta at this stage (green arrow). (D) As Pdx1-positive cells form the dorsal pancreatic bud, dorsal mesenchyme cells (long black arrow) interpose between them and the fused aorta. At this stage, small capillaries can be found in the dorsal mesenchyme (E, arrows). (F,G) At 8S, the prospective ventral foregut endoderm is in the proximity of the sinus venosus. (H,I) At 10S, during embryo turning, the vitelline vein on the embryo's right side is distal to the initial few Pdx1-positive cells in the ventral endoderm. (J-M) A thin line of mesenchyme cells continuously separates the few, nascent Pdx1-positive endoderm cells from the vitelline veins (K,M; cells at end of green arrow). (N) By 26S, when the ventral pancreatic domain is clearly surrounded by mesenchyme cells, small capillaries can be found in the mesenchyme (O, arrow), as seen in the dorsal region. Original magnifications are indicated at the bottom of each panel.
- Fig. 2.
Dorsal pancreatic development is impaired in Flk1-/- embryos after the induction of an initial field of Pdx1-positive cells. Immunostaining of transverse sections of control (upper row) and Flk1-/- (lower row) embryos at E9.0-10.0 (somite stages are indicated on the top of each panel). (A,B) Pecam staining to identify blood vessels; no vessels develop in Flk1-/- embryos. (C-I) Pdx1 staining. Although at 15S, Flk1-/- embryos (D) develop nearly the same size domain of Pdx1-positive cells in the dorsal endoderm as control embryos (C), these cells diminish over time and fail to form a bud (compare G and H). By 31S, very few Pdx1-positive cells can be found in the dorsal endoderm (I). (J) RT-PCR analysis of dorsal midgut RNAs at the designated somite stages (S). The normalizing cycle steps for Pecam and Tal1 were based on prior cycle step analysis of actin. Embryonic aorta served as positive controls. PCR cycle steps: Pecam, 30, 33; Tal1, 30, 33; actin, 26, 29.
- Fig. 3.
Dorsal endoderm of Flk1-/- embryos expresses Foxa2 and Hnf6 and Shh expression is repressed, as in control embryos. In situ hybridization of control (A,C,E) and Flk1-/- (B,D,F) embryos with probes of Foxa2 (A,B), Hnf6 (C,D), and sonic hedgehog (E,F). nc, notochord. Somite stages are indicated in each panel.
- Fig. 4.
Dorsal endoderm in Flk1-/- embryos does not express Ptf1a, insulin and glucagon. (A-C) RT-PCR analysis of the dorsal endoderm in the region of the pancreatic bud. (A) The expression levels of factors involved in early pancreatic development were lower in the dorsal endoderm of Flk1-/- embryos than in that of control embryo. (B) The expression of Ptf1a, insulin, and glucagon was not observed in the dorsal endoderm of Flk1-/- embryos even with three or six additional PCR cycles. PCR cycle ranges of embryo tissues: Pdx1, 35-41; Ptf1a, 34-40; Prox1, 33-39; Ngn3, 34-40; Neurod1, 34-40; insulin, 33-39; glucagon, 27-33; actin, 23-29. (C) The expression of Ptf1a starts by 15S in wild-type embryos, when the aorta has extensive contact with Pdx1-positive cells. Short exposure, 12 hours; long, 5 days. In some experiments, Ptf1a expression was observed in the 12-15 S range (data not shown). PCR cycles for Ptf1a, 39, 42, 45; actin 28, 31, 34. (D,E) Glucagon immunostaining confirms the absence of glucagon expression in the dorsal endoderm of Flk1-/- embryos. Somite stages are indicated in the each panel.
- Fig. 5.
The initiation of the ventral pancreatic development is not affected in Flk1-/- embryos. (A-F) Immunostaining of transverse sections of control (upper row) and Flk1-/- (lower row) embryos at E9.0-10.0 (somite stages are indicated on the top of each panel) with Pecam (A,B) and Pdx1 (C-F) antibodies. No endothelial cells or blood vessels were detected in Flk1-/- embryos (B). Blue arrowhead denotes a possible extraembryonic cell (see text) staining positive for Pecam; these were occasionally detected and were reported by Shalaby et al. (Shalaby et al., 1995). (G) RT-PCR analysis of Pecam and Tal1 expression in ventral midgut/pancreatic bud tissue. Note faint bands in lanes 10 and 12 (Flk1 homozygotes). (H) RT-PCR analysis of the ventral region of control and Flk1-/- embryos. PCR cycle ranges similar to that in Fig. 4A. Note the induction of Ptf1a, but not of glucagon. Insulin is not normally detectable ventrally at these stages (data not shown).
- Fig. 6.
Dorsal aorta fragments induce Ptf1a in dorsal endoderm explants from Flk1-/- embryos. (A-C) Dissection of dorsal endoderm and aorta from E8.5 embryos. (D) RT-PCR analysis of Hnf6 confirms that the gene is expressed in the endoderm fragments and that there is no contamination of endodermal cells in the dissected aorta. PCR cycle ranges for Hnf6, 40-45; actin, 26-30. (E) The endoderm explants were cultured with (upper panels) or without (lower panels) the dorsal aorta for 24 hours and photographed. Marked differences in growth were not observed. (F) Analysis of explant cultures. Hnf6 and Pecam RT-PCR confirm the existence of endoderm and aorta, respectively, in explants, and Hex analysis of the dorsal endoderm excludes the possibility of contamination by ventral pancreas cells (see text). All explants included in the analysis had the Hnf6, Pecam, Hex and actin expression phenotypes shown. For wild-type controls (lanes 5, 6), we used: Ptf1a, adult pancreas; Hnf6, dorsal endoderm from an 8S embryo; Pecam, dorsal aorta from an 8S embryo; Hex, liver and ventral pancreatic buds from a 20S embryo. Four out of five Flk1-/- dorsal endoderm explants cultured with aorta from control embryos expressed Ptf1a, whereas none of the five Hex-negative explants cultured without the aorta expressed Ptf1a. PCR cycle ranges: Ptf1a, 40-43, Hnf6, 39-42; Pecam, 39-42; Hex, 37-40, actin, 28-31. (G) Wt1, a mesenchyme marker (Armstrong et al., 1992), is expressed in the dorsal pancreatic region. PCR cycle ranges: Wt1, 40, 43; actin, 26, 29. (H) Wt1-expressing cells do not persistently contaminate endoderm-aorta explants that induce Ptf1a (RT-PCR analysis). PCR cycle ranges: Ptf1a, 40-44; Wt1, 38-42;
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