Abstract
Blood vessels supply developing organs with metabolic sustenance. Here, we demonstrate a role for blood vessels as a source of developmental signals during pancreatic organogenesis. In vitro experiments with embryonic mouse tissues demonstrate that blood vessel endothelium induces insulin expression in isolated endoderm. Removal of the dorsal aorta in Xenopus laevis embryos results in the failure of insulin expression in vivo. Furthermore, using transgenic mice, we show that ectopic vascularization in the posterior foregut leads to ectopic insulin expression and islet hyperplasia. These results indicate that vessels not only provide metabolic sustenance, but also provide inductive signals for organ development.
Vascular endothelium grows in coordination with all embryonic tissues and is essential for viability (1, 2). Its prevalence in the embryo is likely to be guided by vascular endothelial growth factor (VEGF). VEGF induces proliferation, migration, and differentiation of endothelial cells (3). The main receptors for VEGF—VEGFR1 and VEGFR2 (also known as Flk-1)—are expressed on endothelial cells and their precursors (4). Mice lacking a single VEGF allele die of severe vascular abnormalities (5, 6).
Here, we investigate the role of blood vessels during endocrine pancreatic development. Islets of Langerhans, composed of pancreatic endocrine cells, perform a vital metabolic role in vertebrates that depends on an intimate contact with blood vessels. Islets measure glucose levels in the blood and react by secreting endocrine hormones, predominantly insulin, into the circulatory system (7). In this report, we focus on endothelial induction of insulin gene expression, because insulin is expressed in differentiated endocrine cells and because of its critical role in insulin-dependent diabetes mellitus (IDDM). Unlike other pancreatic hormones, insulin expression is restricted to pancreatic tissue along the gastrointestinal tract.
Pancreatic growth is initiated at three sites in the foregut endoderm, precisely where the endodermal epithelium contacts the endothelium of major blood vessels (Fig. 1, A through F). At this stage, vessels are composed solely of endothelial cells and are not yet surrounded by smooth muscle or other supporting cells. At 9.0 days post coitus (dpc), the pancreatic rudiment is marked by expression of the homeobox genePdx1 (8, 9) which is restricted to endoderm that contacts the endothelium of the vitelline veins and the fusing dorsal aortae (Fig. 1, E and F). Gut endoderm that is either anterior or posterior to the evaginating pancreatic buds is separated from the major blood vessels by mesenchyme at this stage (Fig. 1, A through D). Following endothelial contact, both dorsal and ventral Pdx1domains evaginate (Fig. 1G). At 10.5 dpc, insulin expression begins at sites where the dorsal pancreatic endoderm contacts portal vein endothelium (Fig. 1, H and I) (10, 11).
Pancreatic budding and endocrine differentiation take place adjacent to blood vessels. (A) Schematic representation of blood vessels (red) relative to pancreatic endoderm expressing Pdx1 (blue). The gut tube endoderm is yellow. Aortae (a), vitelline veins (v), and gut endoderm (g) are indicated. (B to D) Transverse sections through mouse foregut, as indicated in (A). Sections are taken from the same 9.5-dpc mouse embryo. (B) Section anterior to the Pdx1 region. (C) Section through the Pdx1 region. (D) Section posterior to the Pdx1 region. (E) In the ventral gut tube (g) at 9 dpc, Pdx1expression begins in two ventral domains. One domain is shown here.Pdx1 is initiated in the endoderm adjacent to the vitelline vein endothelium (v) and not in endoderm adjacent to mesenchyme (m). (F) Pdx1 expression begins slightly later in the dorsal endoderm adjacent to the fusing dorsal aortae (a). (G) These Pdx1-expressing domains are budding from the gut tube at 9.75 dpc. (H and I) Adjacent transverse sections of the mouse gut tube at 10.5 dpc. Scattered cells expressing insulin develop in the dorsal pancreatic bud [arrowheads in (H)] adjacent to the right vitelline or portal vein as marked by VEGFR2 expression [sites of contact with insulin-expressing cells are marked with arrowheads in (I)]. (J through N) Adjacent sections of a neonatal p3 mouse pancreas are stained for insulin (J), VEGF (K), VEGFR2 (L), platelet endothelial-cell adhesion molecule 1 (PECAM1) (M), and duct glycosylation (N). Islets, i; ducts, d. Sections were stained as described (28,29).
The interaction of blood vessels with endocrine cells continues when mature, hormone-secreting islets are formed after birth. As previously observed, islets express significant levels of VEGF (Fig. 1, J and K), specifically the VEGF120 and VEGF164 isoforms, and pancreatic endothelial cells express both VEGF receptors (12). VEGFR2-expressing capillaries surround not only islets but also pancreatic ducts, which may harbor islet precursors (Fig. 1, L through N) (13). Thus, pancreatic-endothelial interactions begin long before islets are functionally mature, and are maintained throughout islet formation, raising the issue of whether these tissues signal to each other during development.
To address signaling between tissues while excluding the effects of embryonic blood flow and metabolic exchange, endoderm and dorsal aortae were isolated from mouse embryos at 8.25 to 8.5 dpc and recombined in culture (Fig. 2 and Table 1) (10). At 8.5 dpc, midline endoderm is associated with the notochord and has not yet formed a tube (Fig. 2, A and B). The endoderm consists of tissue fated to become foregut and midgut, including the dorsal pancreas, but does not yet express pancreatic genes (14). The dorsal aortae lie immediately dorsal to the endoderm as two endothelial tubes, lateral to the notochord (Fig. 2, C and D) (15). Isolated endoderm, grown in culture for 6 days, formed a structure reminiscent of a gut tube, but did not exhibit signs of pancreatic differentiation, such asPdx1 or insulin expression (Fig. 2, E through H). In contrast, when isolated endoderm was cultured in combination with dorsal aortae, it initiated both Pdx1 and insulin expression (Fig. 2, I through L, and Table 1). Approximately one-third of the recombinants formed structures resembling pancreatic buds (Fig. 2I). Insulin expression appeared adjacent to endothelium, as observed during normal development.
Blood vessel endothelium induces insulin expression in isolated mouse endoderm. (A throughD) Dorsal endoderm (arrow) and dorsal aortae (arrowheads) from the first to fifth somite region are isolated from mouse embryos at 8.25 to 8.5 dpc (8–10 somite stage). When cultured alone, the endoderm forms a gut tube–like structure (E), as shown by an H&E (hematoxylin/eosin)–stained section (F). No associated endothelial cells, as assayed by PECAM1 expression (G), and no endocrine cells, as assayed by insulin expression (H), are detected on adjacent sections. (I through L) When the endoderm is recombined with aorta endothelium (I), it forms a tube-like structure as seen by H&E staining (J), exhibits budding (I), and expresses insulin (L) adjacent to aorta endothelium (K).
In vitro recombination of 8.25- to 8.5-dpc mouse tissues demonstrates that endothelial signals induce pancreatic differentiation in isolated endoderm. Tissues used for Pdx1analysis are taken from progeny of matings of mice heterozygous for a lacZ insertion into the Pdx1 locus with ICR mice (half should contain the insertion). Ratios of explants expressing insulin (as determined by antibody staining) and Pdx1 (as determined by LacZ staining) versus total number of explants are shown. n.d., not done.
Recombination of prepancreatic endoderm with other tissues harboring endothelial cells, including the lateral plate mesenchyme and umbilical artery, also lead to pancreatic differentiation (Table 1) (10). In contrast, tissues such as the notochord or neural tube did not induce insulin expression in isolated endoderm. The notochord induced Pdx1, but never insulin, in these mouse endodermal explants (Table 1). Our previous work with chick embryos showed that the notochord induced bothPdx1 and insulin [(16); for discussion, see (17)]. To demonstrate that the isolated aortae did not carry along residual insulin cell precursors, we recombined dorsal aortae with other tissues such as somites and neural tube, and yet insulin expression was never observed in these recombinants (Table 1). These results indicate that insulin expression is induced in competent prepancreatic endoderm and that endothelium supplies signals sufficient for insulin expression.
To assess the requirement for endothelial signals during endocrine cell differentiation in vivo, we generated Xenopusembryos lacking a dorsal aorta (18) and examined the consequences on pancreatic development (19). The expression of insulin and two endocrine transcription factors, NeuroD(20) and Pax6 (21), marks the dorsal pancreatic anlage in Xenopus (22). Embryos lacking a dorsal aorta displayed a strong reduction or elimination of insulin, NeuroD, and Pax6 expression in all dissected embryos (Fig. 3, A through F) (10). These results indicate that endothelial signals are required for endocrine cell differentiation in frog embryos.
(A through F) The dorsal aorta is necessary for endocrine pancreatic differentiation in stage 39–40 Xenopus embryos. Immunohistochemistry for insulin and in situ hybridization for NeuroD and Pax6 in the pancreatic anlage of unmanipulated embryos (A to C) and dissected embryos (D to F). (G through L) In situ hybridization for F-spondin in the floor plate and hypochord, and XHex in the liver, indicates that midline development of mesoderm and endoderm can proceed normally in the absence of the dorsal aorta, subsequent to the dissection procedure. Notochord (n), aorta (a), and endoderm (e) are indicated. (G and J) H&E-stained sections show the absence of the dorsal aorta in dissected embryos.
Blocking aorta formation is accomplished by excising intermediate mesoderm, which contains aortic precursors (18). Controls demonstrated that the dissection procedure did not adversely affect tissues located nearby, including the notochord, hypochord, gut tube, and floor plate. This was shown by normal notochord development (Fig. 3, G and J), the normal expression patterns for F-spondin (23) in the hypochord and floor plate (Fig. 3, H and K), and XHex expression (24) in liver and thyroid endoderm (Fig. 3, I and L). We note that Xenopus embryos did not express endocrine pancreatic genes, despite the presence of the notochord, when aorta formation was prevented. Moreover, the absence of the intermediate mesoderm by itself did not result in failure of endocrine development, because embryos lacking intermediate mesoderm expressed insulin when endothelium was present (10). Thus, endocrine pancreatic gene expression in Xenopus invariably correlates with the presence of endothelial tissue.
To assay blood vessel–pancreas interactions later in development, we constructed a mouse transgene in whichVEGF164, coexpressed with EGFP (enhanced green fluorescent protein), is driven by the Pdx1 promoter (10, 25). During murine embryogenesis, Pdx1 is expressed throughout the pancreas and parts of the stomach and duodenum (see below), and becomes progressively restricted to islets by birth (9, 26). Animals with strong VEGF expression could therefore be selected by the presence of green fluorescence in the dorsal pancreas (Fig. 4A). As a result of VEGF expression (10), fluorescent Pdx-VEGFtransgenic animals displayed a marked increase in vascular endothelium (compare Fig. 4B with 4C).
Hypervascularization of the pancreas results in islet hyperplasia. (A) Expression of thePdx-VEGF transgene in 15.5-dpc embryonic pancreas is assayed by EGFP fluorescence in the pancreas. (B) Transgenic embryos display hypervascularization of pancreatic tissue, as assayed by PECAM1 when compared with (C) nontransgenic controls. (Dthrough I) Islet hyperplasia in the pancreas of transgenic animals at 2 months. Adjacent pancreas sections fromPdx-VEGF transgenic mice (G through I) are compared with nontransgenic littermates (D through F). Sections are stained for insulin (D and G), pancreatic polypeptide PP (E and H), and PECAM1 (F and I).
At 2 months of age, Pdx-VEGF transgenic mice had a cystic, hypervascularized pancreas and islet hyperplasia (compareFig. 4, D through F, and Fig. 4, G through I). Pdx-VEGFtransgenic mice displayed a threefold increase in islet number and islet area, accompanied by a sevenfold decrease in the acini area (10, 27). Evidently, endocrine cell types were promoted at the expense of acinar cell types. Transgenic islets revealed a normal islet morphology (Fig. 4, G and H) (10).
Previous work (8, 9) had shown that after its initial localization in pancreatic buds at 9.5 dpc, Pdx1expression expands to the posterior part of the stomach and to the duodenum by 11.5 dpc (Fig. 5A). We therefore examined the stomach and duodenum of Pdx-VEGFtransgenic embryos for developmental effects in transient transgenic experiments. At 15.5 dpc, five of five transgenic embryos with strong fluorescence had ectopic insulin-expressing cells in the posterior stomach epithelium (Fig. 5, B and H). Insulin expression was not detected in the stomach of nontransgenic littermates (0 of 20, Fig. 5E). A comparison of transgenic and nontransgenic littermates demonstrated that ectopic insulin-expressing cells were found directly adjacent to ectopically induced endothelial cells (compare Fig. 5, D and E, with Fig. 5, G and H). These cells were located within thePdx1-expressing posterior stomach epithelium. Insulin-expressing cells were found neither in the hypervascularized stomach mesenchyme nor in the epithelium outside the Pdx1region (Fig. 5, B and H). Thus, it is likely that the ectopic insulin-positive cells developed from Pdx1-expressing precursor cells within the stomach epithelium. The degree of ectopic insulin expression in the epithelium correlated directly with the degree of hypervascularization (10). In two of five transgenic animals, an ectopic budlike structure was observed in the anterior duodenum (10). The ectopic bud had particularly high levels of VEGF transgene expression and scattered insulin-expressing cells.
Ectopic vascularization leads to ectopic insulin expression. (A) Pdx1expression as detected by a lacZ gene inserted into the Pdx1locus (9). Cleared embryos at 9.5, 10.5, and 11.5 dpc show that Pdx1 expression expands beyond the dorsal (dp) and ventral (vp) pancreatic buds, to the duodenum (duo) and stomach (sto) during embryonic development. (B) Staining for insulin (red) and DAPI nuclei (blue) staining on a 5-μm stomach section of a 15.5-dpc Pdx-VEGF transgenic mouse. Adjacent stomach sections of transgenic mice (F through H) are compared with nontransgenic littermates (C throughE). Sections are stained for VEGF (C and F), VEGFR2 (D and G), and insulin (E and H).
In this study, we have used three different experimental systems to demonstrate a role for blood vessels as a source for developmental signals during pancreatic organogenesis. Specifically, we showed that blood vessel endothelium induces endocrine pancreatic differentiation. Taken together with previous embryological work (14, 16), these data indicate that prepancreatic endoderm is patterned in a stepwise manner, beginning with signals from the mesoderm/ectoderm at 7.5 dpc (14), followed by signals from the notochord during the next day (16). The blood vessel endothelium then provides the prepatterned endoderm with additional signals that induce differentiation of insulin-expressing cells. The fact that early endoderm, including the prepancreatic region, expresses VEGF (4) could explain how the vessels are attracted to the growing pancreas and islets. In a broader context, our data suggest that the close physiological relationship between blood vessels and islets in the pancreas depends on inductive interactions that begin at the earliest stages of development. This type of mutual signaling between tissues and blood vessels during development may be a general mechanism used in vertebrate organogenesis.
↵* These authors contributed equally to this work.
