During pregnancy, the energy requirements of the fetus impose changes in maternal metabolism. Increasing insulin resistance in the mother maintains nutrient flow to the growing fetus, while prolactin and placental lactogen counterbalance this resistance and prevent maternal hyperglycemia by driving expansion of the maternal population of insulin-producing β-cells1–3. However, the exact mechanisms by which the lactogenic hormones drive β-cell expansion remain uncertain. Here we show that serotonin acts downstream of lactogen signaling to drive β-cell proliferation. Serotonin synthetic enzyme Tph1 and serotonin production increased sharply in β-cells during pregnancy or after treatment with lactogens in vitro. Inhibition of serotonin synthesis by dietary tryptophan restriction or Tph inhibition blocked β-cell expansion and induced glucose intolerance in pregnant mice without affecting insulin sensitivity. Expression of the Gαq-linked serotonin receptor Htr2b in maternal islets increased during pregnancy and normalized just prior to parturition, while expression of the Gαi-linked receptor Htr1d increased at the end of pregnancy and postpartum. Blocking Htr2b signaling in pregnant mice also blocked β-cell expansion and caused glucose intolerance. These studies reveal an integrated signaling pathway linking β-cell mass to anticipated insulin need during pregnancy. Modulators of this pathway, including medications and diet, may affect the risk of gestational diabetes4.

Insulin from the β-cells of the pancreatic islets of Langerhans controls energy homeostasis in vertebrates, and its deficiency causes diabetes mellitus. During embryonic development, the transcription factor Neurogenin3 initiates the differentiation of the β-cells and other islet cell types from pancreatic endoderm, but the genetic program that subsequently completes this differentiation remains incompletely understood. Here we show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurogenin3. Mice lacking Rfx6 failed to generate any of the normal islet cell types except for pancreatic-polypeptide-producing cells. In human infants with a similar autosomal recessive syndrome of neonatal diabetes, genetic mapping and subsequent sequencing identified mutations in the human RFX6 gene. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors that coordinate pancreatic islet development in both mice and humans. Rfx6 could prove useful in efforts to generate β-cells for patients with diabetes.

The distal portion of the rat insulin I gene 5'-flanking DNA contains two sequence elements, the Far and FLAT elements, that can function in combination, but not separately, as a 13-cell-specific transcriptional enhancer. We have isolated several cDNAs encoding proteins that bind to the FLAT element. Two of these cDNAs, cdx-3 and Imx-1, represent homeo box containing mRNAs with restricted patterns of expression. The protein encoded by lmx-1 also contains two amino-terminal cysteine/histidine-rich "LIM" domains. Both cdx-3 and Imx-1 can activate transcription of a Far/FLAT-linked gene when expressed in a normally non-insulin-producing fibroblast cell line. Furthermore, in fibroblasts expressing transfected 13-cell lmx-1, the addition of the Far-binding, basic helix-loop-helix protein shPan-1 (the hamster equivalent of human E47) causes a dramatic synergistic activation. ShPan-1 causes no activation in fibroblasts expressing transfected cdx-3 or the related LIM-homeodomain protein isl-1. Deletion of one or both of the LIM domains from the 5' end of the lmx-1 cDNA removes this synergistic interaction with shPan-1 without any loss of basal transcriptional activation. We conclude that 13-cell lmx-1 functions by binding to the FLAT element and interacting through the LIM-containing amino terminus with shPan-1 bound at the Far element. These proteins form the minimal components for a functional minienhancer complex.

Neurogenin3 (ngn3), a basic helix-loop-helix (bHLH) transcription factor, functions as a pro-endocrine factor in the developing pancreas: by itself, it is sufficient to force undifferentiated pancreatic epithelial cells to become islet cells. Because ngn3 expression determines which precursor cells will differentiate into islet cells, the signals that regulate ngn3 expression control islet cell formation. To investigate the factors that control ngn3 gene expression, we mapped the human and mouse ngn3 promoters and delineated transcriptionally active sequences within the human promoter. Surprisingly, the human ngn3 promoter drives transcription in all cell lines tested, including fibroblast cell lines. In contrast, in transgenic animals the promoter drives expression specifically in regions of ngn3 expression in the developing pancreas and gut; and the addition of distal sequences greatly enhances transgene expression. Within the distal enhancer, binding sites for several pancreatic transcription factors, including hepatocyte nuclear factor (HNF)-1 and HNF-3, form a tight cluster. HES1, an inhibitory bHLH factor activated by Notch signaling, binds to the proximal promoter and specifically blocks promoter activity. Together with previous genetic data, these results suggest a model in which the ngn3 gene is activated by the coordinated activities of several pancreatic transcription factors and inhibited by Notch signaling through HES1. Diabetes 50:928 -936, 2001

Like expression of other cell-type-specific genes, expression of the insulin gene depends on the actions of a unique set of nuclear activators. These activators cooperate synergistically in building a transcriptional activation complex that binds to the regulatory domains of the gene and activates the basal RNA polymerase machinery (reviewed in reference 7). The complexity and specificity of the interactions among these activators limit the cell types capable of building a functional activation complex. Dissection of these interactions provides insight into the mechanism by which insulin expression is limited to the correct cell type.In adult mammals, activation of the insulin gene is tightly restricted to the ␤ cells in the pancreatic islets of Langerhans, where it is expressed at high levels. This specificity is reflected in the restricted function of the insulin promoter, the proximal few hundred base pairs of which can replicate the specificity of the intact gene (19,50). Because of the complexity of the intact promoter (9, 13, 24), a short portion of the rat insulin I promoter between bp Ϫ247 and Ϫ197 upstream from the transcription initiation site has been used as a model of the types of synergistic interactions that combine to give the characteristic activity of the full promoter (15). This 50-bp fragment contains at least three distinct DNA-binding sites named E2, A3, and A4 (13). The E and A elements synergize: neither has significant activity on its own, but in combination E and A elements produce ␤-cell specific transcriptional activation (15,23).The E2 element functions as a recognition site for dimers of basic helix-loop-helix (bHLH) proteins, including a heterodimer of the ubiquitous bHLH protein E47/Pan1 and the neuroendocrine specific bHLH protein BETA2/NeuroD1 (35). The A elements each contain the sequence TAAT and have been shown to bind to several homeodomain proteins found in ␤-cell nuclei (12,16,22, 32,37 (16,38,40).The LIM homeodomain protein Lmx1.1 contains two LIM domains that form zinc-binding structures in the amino end of the molecule. The second of these two LIM domains (LIM2) directly binds to the bHLH domain of E47/Pan1 and mediates the synergy between Lmx1.1 and E47/Pan1. This interaction is specific, since analogous domains from other LIM proteins and bHLH proteins cannot substitute for the LIM2 domain of Lmx1.1 or the bHLH domain of E47/Pan1, respectively (20).PDX-1 plays an important role both in the development of the pancreas and in maintaining ␤-cell function. Mice with a targeted disruption of the pdx1 gene selectively lack a pancreas (2, 21, 36). Similar pancreatic agenesis has been found in a human patient with a single nucleotide deletion in the pdx1 gene (46). If the pancreas is allowed to develop with an intact pdx1 gene, and the pdx1 gene is disrupted only in mature ␤ cells, diabetes ensues due to impaired ␤-cell function (3). This

OBJECTIVEDespite their origins in different germ layers, pancreatic islet cells share many common developmental features with neurons, especially serotonin-producing neurons in the hindbrain. Therefore, we tested whether these developmental parallels have functional consequences.RESEARCH DESIGN AND METHODSWe used transcriptional profiling, immunohistochemistry, DNA-binding analyses, and mouse genetic models to assess the expression and function of key serotonergic genes in the pancreas.RESULTSWe found that islet cells expressed the genes encoding all of the products necessary for synthesizing, packaging, and secreting serotonin, including both isoforms of the serotonin synthetic enzyme tryptophan hydroxylase and the archetypal serotonergic transcription factor Pet1. As in serotonergic neurons, Pet1 expression in islets required homeodomain transcription factor Nkx2.2 but not Nkx6.1. In β-cells, Pet1 bound to the serotonergic genes but also to a conserved insulin gene regulatory element. Mice lacking Pet1 displayed reduced insulin production and secretion and impaired glucose tolerance.CONCLUSIONSThese studies demonstrate that a common transcriptional cascade drives the differentiation of β-cells and serotonergic neurons and imparts the shared ability to produce serotonin. The interrelated biology of these two cell types has important implications for the pathology and treatment of diabetes.

In the developing pancreas, the basic helix-loop-helix (bHLH) protein Neurogenin3 (Ngn3) specifies which precursor cells ultimately will become endocrine cells and initiates the islet differentiation program. NeuroD1, a closely related bHLH protein and a downstream target of Ngn3, maintains the differentiation program initiated by Ngn3. We have developed an in vitro model of Ngn3-dependent differentiation by infecting pancreatic duct cell lines with an Ngn3-expressing adenovirus. We found that both Ngn3 and its downstream target NeuroD1 activated the islet differentiation program in these cells by inducing the expression of genes with early roles in the differentiation cascade, as well as genes characteristic of fully differentiated islet cells. Induction of these genes, as exemplified by the insulin1 gene, involved alteration of the local chromatin structure. Interestingly, the subsets of genes activated by Ngn3 and NeuroD1 were not completely overlapping, indicating that these two bHLH proteins serve specific functions in the development of the endocrine pancreas. In addition, microarray gene expression analysis identified a previously uncharacterized group of Ngn3-induced genes with potentially important roles in islet development and function. These studies demonstrate how Ngn3 initiates islet differentiation and provide us with a model for testing methods for producing islet cells for people with diabetes. During pancreatic development, differentiation of endocrine and exocrine cells from a common endodermal progenitor cell requires the coordinated regulation of specific sets of genes. This process can be envisioned as a hierarchy or cascade of transcription factors that initiate and maintain the distinct gene expression programs that define the various pancreatic cell types (1). Among these factors, the basic helix-loop-helix (bHLH) protein Neurogenin3 (Ngn3) plays a dominant role in the specification of the endocrine͞islet cell lineage.During embryonic development, Ngn3 appears transiently in scattered pancreatic epithelial cells (2, 3). Several lines of evidence indicate that the expression of Ngn3 in these undifferentiated cells directs them to an endocrine cell fate and initiates the program of islet differentiation. First, lineage tracing shows that these transient Ngn3-expressing cells differentiate exclusively into islet cells (4). Second, mice homozygous for a targeted deletion of the ngn3 gene fail to generate any islet cells (5). Third, ectopic expression of Ngn3 drives embryonic endoderm to an endocrine fate (2, 3, 6).Ngn3 may play a similar role in the generation of new islet cells postnatally. It has been suggested that cells along the pancreatic ducts may act as progenitors for new islet cells in the postnatal period, although recent lineage tracing experiments suggest that the bulk of newly generated beta cells in adult mice result from the replication of preexisting beta cells (7).Ngn3 initiates islet cell differentiation, but other factors downstream of ngn3 must complete the task. Genetic...

To investigate the role of the Sry/hydroxymethylglutaryl box (Sox) transcription factors in the development of the pancreas, we determined the expression pattern of Sox factors in the developing mouse pancreas. By RT-PCR, we detected the presence of multiple Sox family members in both the developing pancreas and mature islets and then focused on two factors, Sox2 and Sox4. The expression field of Sox2, which plays a role in the maintenance of some stem cell populations, included the developing duodenum, but Sox2 was specifically excluded from the pancreatic buds. In contrast, Sox4 was detected broadly in the early pancreatic buds and eventually became restricted to the nuclei of all islet cells in the adult mouse. Mice homozygous for a null mutation of the sox4 gene showed normal pancreatic bud formation and endocrine cell differentiation up to embryonic day 12.5. Beyond that date, cultured pancreatic explants lacking sox4 failed to form normal islets. Instead, a markedly reduced number of endocrine cells were found scattered through the explant. We show here that several Sox transcription factors are expressed in the developing pancreas and in the islet, and that one of these factors, Sox4, is required for the normal development of pancreatic islets. Diabetes 54:3402-3409, 2005 S cattered through the exocrine pancreas, the islets of Langerhans are highly organized clusters of endocrine cells comprised of four distinct cell types: the glucagon-secreting ␣-cells, the insulinsecreting ␤-cells, the somatostatin-secreting ␦-cells, and the pancreatic polypeptide-secreting cells. Because of the essential role of the islet hormones in energy metabolism, defects in the development, maintenance, or function of the endocrine pancreas have serious consequences, including diabetes.The pancreas forms from the endoderm in the region of the foregut/midgut junction and is first visible in mice at embryonic day 9.5 (e9.5) (1). The first endocrine cells of the pancreas appear at around e9.5 in the dorsal pancreatic bud. These cells express glucagon; a few insulinexpressing cells appear ϳ1 day later. At ϳe13-14, the pancreas undergoes a distinct change termed the secondary transition, characterized by the appearance of ductal cells, exocrine cells, and delta cells and a rapid expansion of the insulin-expressing cells. The endocrine cells found in the pancreas before the secondary transition have clear differences from mature islet cells and may result from developmental pathways that are distinct from those that produce the more mature endocrine cells that arise following the secondary transition (2-6). By e18, the first pancreatic polypeptide-expressing cells appear, and the endocrine cells organize into distinct islets with the ␤-cells forming the central core. Around the same time, ␤-cell neogenesis declines, but the simultaneous onset of ␤-cell replication continues to expand the ␤-cell population and enlarge the forming islets (7).This process of endocrine cell determination and differentiation depends on the proper sequentia...