Pancreas morphogenesis and cell differentiation are highly conserved among vertebrates during fetal development. The pancreas develops through simple budlike structures on the primitive gut tube to a highly branched organ containing many specialized cell types. This review presents an overview of key molecular components and important signaling sources illustrated by an extensive three-dimensional (3D) imaging of the developing mouse pancreas at single cell resolution. The 3D documentation covers the time window between embryonic days 8.5 and 14.5 in which all the pancreatic cell types become specified and therefore includes gene expression patterns of pancreatic endocrine hormones, exocrine gene products, and essential transcription factors. The 3D perspective provides valuable insight into how a complex organ like the pancreas is formed and a perception of ventral and dorsal pancreatic growth that is otherwise difficult to uncover. We further discuss how this global analysis of the developing pancreas confirms and extends previous studies, and we envisage that this type of analysis can be instrumental for evaluating mutant phenotypes in the future.

BackgroundEndoderm organ primordia become specified between gastrulation and gut tube folding in Amniotes. Although the requirement for RA signaling for the development of a few individual endoderm organs has been established a systematic assessment of its activity along the entire antero-posterior axis has not been performed in this germ layer.Methodology/Principal FindingsRA is synthesized from gastrulation to somitogenesis in the mesoderm that is close to the developing gut tube. In the branchial arch region specific levels of RA signaling control organ boundaries. The most anterior endoderm forming the thyroid gland is specified in the absence of RA signaling. Increasing RA in anterior branchial arches results in thyroid primordium repression and the induction of more posterior markers such as branchial arch Hox genes. Conversely reducing RA signaling shifts Hox genes posteriorly in endoderm. These results imply that RA acts as a caudalizing factor in a graded manner in pharyngeal endoderm. Posterior foregut and midgut organ primordia also require RA, but exposing endoderm to additional RA is not sufficient to expand these primordia anteriorly. We show that in chick, in contrast to non-Amniotes, RA signaling is not only necessary during gastrulation, but also throughout gut tube folding during somitogenesis. Our results show that the induction of CdxA, a midgut marker, and pancreas induction require direct RA signaling in endoderm. Moreover, communication between CdxA + cells is necessary to maintain CdxA expression, therefore synchronizing the cells of the midgut primordium. We further show that the RA pathway acts synergistically with FGF4 in endoderm patterning rather than mediating FGF4 activity.Conclusions/SignificanceOur work establishes that retinoic acid (RA) signaling coordinates the position of different endoderm organs along the antero-posterior axis in chick embryos and could serve as a basis for the differentiation of specific endodermal organs from ES cells.

Ptf1a+ and amylase + cells, occupying the proximal domain, suggests that proximal cells adopt a distal fate in the absence of Mib1 activity. Impeding Notch-mediated transcriptional activation by conditional expression of dominant negative Mastermind-like 1 (Maml1) resulted in a similarly distorted P-D patterning and suppressed β-cell formation, as did conditional inactivation of the Notch target gene Hes1. Our results reveal iterative use of Notch in pancreatic development to ensure correct P-D patterning and adequate β-cell formation.diabetes | lateral signaling | tip | trunk

Insulin promoter factor 1 (IPF1), a member of the homeodomain protein family, serves an early role in pancreas formation, as evidenced by the lack of pancreas formation in mice carrying a targeted disruption of the IPF1 gene [Jonsson, J., Carlsson, L., Edlund, T. & Edlund, H. (1994) Nature (London) 371,[606][607][608][609]. In adults, IPF1 expression is restricted to the a-cells in the islets of Langerhans. We report here that IPF1 induces expression of a subset of 8-cell-specific genes (insulin and islet amyloid polypeptide) when ectopically expressed in clones of transformed pancreatic islet a-cells. In contrast, expression of IPF1 in rat embryo fibroblasts factor failed to induce insulin and islet amyloid polypeptide expression. This is most likely due to the lack of at least one other essential insulin gene transcription factor, the basic helix-loop-helix protein Beta2/NeuroD, which is expressed in both a-and P-cells. We conclude that IPF1 is a potent transcriptional activator of endogenous insulin genes in non-8 islet cells, which suggests an important role of IPF1 in a-cell maturation.Insulin promoter factor 1 (IPF1) is expressed in precursor cells during pancreas ontogeny (1, 2), and expression is required for pancreas formation (3,4). During ontogeny, IPF1 expression becomes restricted to the nuclei of the insulin-producing pancreatic islet (3-cells, suggesting that maintenance of IPF1 expression is necessary for the differentiation islet p3-cells from an IPF1-positive precursor common to all islet cells (2, 5). This restricted expression profile within the islets is reflected in the transplantable rat pancreatic insulinoma (IN) and glucagonoma (AN), which show substantial similarity to the mature islet f3-and a-cells, respectively (6-8). Thus, the AN is lacking IPF1 expression, as is the normal a-cell, and was recently found to be similar to normal a-cells in its expression of glucokinase as well as of the glucose-regulated insulinotropic peptide and glucagon-like peptide 1 receptors (9, 10). In vitro IPF1 binds to multiple sites in the insulin promoter and activates insulin gene reporter constructs when cotransfected into cell lines (1,5,11,12). This activity is dependent on cooperation between IPF1 and insulin enhancer factor-1 (IEF-1; refs. 5 and 12), a heterodimer composed of Beta2/ NeuroD, which is present in both a-and (3-cells, and ubiquitous class A helix-loop-helix proteins, such as Betal/rat E-box binding protein (REB; ref. 13) and products of the E2A gene (E47, E12, and ITF-1; refs. 14-19). In addition to IPF1 and IEF-1 binding sites, transcriptional regulation of the insulin gene requires a number of other cis-elements to which factors not yet cloned are binding (20)(21)(22)(23). To address whether IPF1 could activate transcription of the otherwise silent insulin genes in islet cells lacking IPF1 but expressing at least a subset of the other insulin gene transcription factors, a cDNA encoding rat IPF1 (24) under transcriptional control of the cytomegalovirus promoter was stably tra...

Nkx family members are essential for normal development of many different tissues such as the heart, lungs, thyroid, prostate, and CNS. Here, we describe the endodermal expression pattern of three Nkx6 family genes of which two shows conserved expression in the early pancreatic epithelium. In chicken, Nkx6.1 expression is not restricted to the presumptive pancreatic area but is more broadly expressed in the endoderm. In mice, expression of Nkx6.1 is restricted to the pancreatic epithelium. In both mice and chicken, Nkx6.2 and Pdx1 are expressed in very similar domains, identifying Nkx6.2 as a novel marker of pancreas endoderm. Additionally, our results show that Nkx6.3 is expressed transiently in pancreatic endoderm in chicken but not mouse embryos. At later stages, Nkx6.3 is found in the caudal stomach and rostral duodenum in both species. Finally, we demonstrate that Pdx1 is required for Nkx6.1 but not Nkx6.2 expression in mice and that ectopic Pdx1 can induce Nkx6.1 but not Nkx6.2 or Nkx6.3 expression in anterior chicken endoderm. These results demonstrate that Nkx6.1 lies downstream of Pdx1 in a genetic pathway and that Pdx1 is required and sufficient for Nkx6.1 expression in the early foregut endoderm.