The mammalian embryo represents a fundamental paradox in biology. Its location within the uterus, especially early during development when embryonic cardiovascular development and placental blood flow are not well-established, leads to an obligate hypoxic environment. Despite this hypoxia, the embryonic cells are able to undergo remarkable growth, morphogenesis, and differentiation. Recent evidence suggests that embryonic organ differentiation, including pancreatic β-cells, is tightly regulated by oxygen levels. Since a major determinant of oxygen tension in mammalian embryos after implantation is embryonic blood flow, here we used a novel survivable in utero intracardiac injection technique to deliver a vascular tracer to living mouse embryos. Once injected, the embryonic heart could be visualized to continue contracting normally, thereby distributing the tracer specifically to only those regions where embryonic blood was flowing. We found that the embryonic pancreas early in development shows a remarkable paucity of blood flow, and that the presence of blood flow correlates with the differentiation state of the developing pancreatic epithelial cells in the region of the blood flow.

The pancreatic islets of Langerhans are highly vascularized structures scattered throughout the pancreas that contain a capillary network 5-10 times denser than that of the exocrine pancreas. A simple method for three-dimensional (3D) analysis of this intricate intraislet vasculature has been difficult because of the intrinsic opacity of the pancreas. We developed a whole-mount imaging technique that allows relatively easy visualization of the islet vasculature. In combination with confocal microscopy and the use of 3D imaging software, we were able to readily reconstruct the 3D architecture of an islet, allowing delineation of the islet volume, length of the intraislet vessels, and the number of vessel branch-points. This technique allows for straightforward 3D image analysis that may help toward understanding islet function.

Congenital diaphragmatic hernia (CDH) is associated with significant mortality due to lung hypoplasia and pulmonary hypertension. The role of embryonic pulmonary innervation in normal lung development and lung maldevelopment in CDH has not been defined. We hypothesize that developmental defects of intrapulmonary innervation, in particular autonomic innervation, occur in CDH. This abnormal embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH. To define patterns of pulmonary innervation in CDH, human CDH and control lung autopsy specimens were stained with the pan-neural marker S-100. To further characterize patterns of overall and autonomic pulmonary innervation during lung development in CDH, the murine nitrofen model of CDH was utilized. Immunostaining for protein gene product 9.5 (a pan-neuronal marker), tyrosine hydroxylase (a sympathetic marker), vesicular acetylcholine transporter (a parasympathetic marker), or VIP (a parasympathetic marker) was performed on lung whole mounts and analyzed via confocal microscopy and three-dimensional reconstruction. Peribronchial and perivascular neuronal staining pattern is less complex in human CDH than control lung. In mice, protein gene product 9.5 staining reveals less complex neuronal branching and decreased neural tissue in nitrofen-treated lungs from embryonic day 12.5 to 16.5 compared with controls. Furthermore, nitrofen-treated embryonic lungs exhibited altered autonomic innervation, with a relative increase in sympathetic nerve staining and a decrease in parasympathetic nerve staining compared with controls. These results suggest a primary defect in pulmonary neural developmental in CDH, resulting in less complex neural innervation and autonomic imbalance. Defective embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH.

OBJECTIVE-Glucose-dependent insulinotropic polypeptide (GIP) is a member of a structurally related group of hormones that also includes glucagon, glucagon-like peptides, and secretin. GIP is an incretin, known to modulate glucose-induced insulin secretion. Recent studies have shown that glucagon is necessary for early insulin-positive differentiation, and a similar role for incretins in regulating embryonic insulin-positive differentiation seems probable. Here we studied the role of GIP signaling in insulin-positive differentiation in the embryonic mouse pancreas.RESEARCH DESIGN AND METHODS-The ontogeny of the GIP ligand and GIP receptor in the embryonic pancreas was investigated by immunohistochemistry and RT-PCR. GIP signaling was inhibited in cultured embryonic pancreata using morpholinering antisense against GIP ligand and receptor, or small interfering RNA (siRNA) for GIP ligand and receptor. Markers of endocrine cells and their progenitors were studied by immunohistochemistry and RT-PCR.RESULTS-GIP and GIP receptor mRNA were both detected in the embryonic pancreas by embryonic day 9.5 and then persisted throughout gestation. GIP was generally coexpressed with glucagon by immunostaining. The GIP receptor was typically coexpressed with insulin. Morpholine-ring antisense or siRNA against either GIP ligand or GIP receptor both inhibited the differentiation of insulin-positive cells. Inhibition of GIP or its receptor also led to a decrease in the number of Pdx-1-positive and sox9-positive cells in the cultured embryonic pancreas. The number of Pax6-and Nkx2.2-positive cells, representative of developing pancreatic endocrine cells and b-cells, respectively, was also decreased.CONCLUSIONS-GIP signaling may play a role in early embryonic pancreas differentiation to form insulin-positive cells or b-cells. Diabetes 60:548-554, 2011 G lucose-dependent insulinotropic polypeptide (GIP) is an incretin. Incretins are hormones released from the gut in response to nutrient ingestion that potentiate glucose-stimulated insulin secretion (1). GIP and glucagon-like peptide 1 (GLP-1) are the two main incretins, identified as mediators of the process by which administration of oral glucose provokes a greater stimulation of insulin release than does an intravenous glucose challenge (2). This connection between gut and the pancreatic islets has been termed the "enteroinsular axis" (3) and appears to be responsible for 50% of postprandial insulin release (2,4). GIP is released from enteroendocrine K-cells in the duodenum, primarily in response to the ingestion of glucose or fat, and potentiates insulin secretion in a glucose-dependent manner (5). A recent study now reports the presence of a bioactive form of GIP in the a-cells that promotes insulin secretion in the adult islet b-cells (6). GLP-1 is a proglucagon-derived peptide hormone that is synthesized and secreted by the enteroendocrine L-cells in the distal ileum and the colon. Preproglucagon can be alternatively processed to produce glicentin or oxyntomodulin and GLP-1 (7).Th...