Summary The molecular mechanisms that underlie cell lineage diversification of multipotent progenitors in the pancreas are virtually unknown. Here, we show that the early fate choice of pancreatic progenitors between the endocrine and acinar cell lineage is restricted by cross-repressive interactions between the transcription factors Nkx6.1/Nkx6.2 (Nkx6) and Ptf1a. Using genetic loss- and gain-of-function approaches, we demonstrate that Nkx6 factors and Ptf1a are required and sufficient to repress the alternative lineage program and to specify progenitors toward an endocrine or acinar fate, respectively. The Nkx6/Ptf1a switch only operates during a critical competence window when progenitors are still multipotent and can be uncoupled from cell differentiation. Thus, cross-antagonism between Nkx6 and Ptf1a in multipotent progenitors governs the equilibrium between endocrine and acinar cell neogenesis required for normal pancreas development.

SummaryNKX6 transcription factor activity is required for α-and β-cell development in the pancreas

DEVELOPMENT 2491 RESEARCH ARTICLE INTRODUCTIONRecent studies suggest that the most promising approach for the in vitro derivation of insulin-producing pancreatic beta-cells from stem cells is by recapitulating embryonic beta-cell differentiation (D'Amour et al., 2006). The in vitro generation of fully functional beta-cells for transplantation, however, will require further improvement of existing differentiation protocols. Such refinement will require detailed knowledge of the molecular mechanisms that underlie beta-cell differentiation. Lineage-tracing studies in mice have shown that all pancreatic lineages, which include the exocrine and endocrine compartment, arise from a common, proliferative progenitor cell population that is marked by the transcription factor Pdx1 (Gu et al., 2002). Expression of the transcription factor Ngn3 (also known as Neurog3 -Mouse Genome Informatics) further restricts progenitors to five distinct endocrine cell fates: alpha-, beta-, delta-, PP-or epsilon-cells, which produce the hormones glucagon, insulin, somatostatin, pancreatic polypeptide and ghrelin, respectively (Gu et al., 2002;Heller et al., 2005;Prado et al., 2004). Initially, scattered endocrine cells differentiate throughout the organ, but these cells aggregate into so-called islets of Langerhans at the end of gestation (Slack, 1995).In recent years, major progress has been made in our understanding of the molecular pathways that control endocrine differentiation (Jensen, 2004). Much of this knowledge stems from genetic gain-and loss-of-function experiments in mice. Such studies have shown that Ngn3 activity is essential for the differentiation of all endocrine cells and, conversely, that Ngn3 is sufficient to restrict Pdx1-positive (Pdx1 + ) progenitors to an endocrine fate (Apelqvist et al., 1999;Gradwohl et al., 2000;Schwitzgebel et al., 2000). Based on the observation that only select endocrine lineages are affected in null mutant mice for Nkx2.2 (Nkx2-2), NeuroD (Neurod1), Pax4, Arx, Hb9 (also known as Hlxb9 -Mouse Genome Informatics) or Nkx6.1 (Nkx6-1), these transcription factors have been proposed to be downstream effectors of Ngn3 (Collombat et al., 2003;Harrison et al., 1999;Li et al., 1999;Naya et al., 1997;Sander et al., 2000b;Schwitzgebel, 2001;Sosa-Pineda et al., 1997;Sussel et al., 1998;Wilson et al., 2003). Although the loss of Pax4, Arx and NeuroD expression in Ngn3 mutant mice (Collombat et al., 2003;Gradwohl et al., 2000) indeed suggests a function of these genes downstream of Ngn3 in endocrine differentiation, the evidence for Nkx6.1 being downstream of Ngn3 is less clear.Deficiency for Nkx6.1 results in a specific abrogation of beta-cell neogenesis during embryogenesis without affecting cell survival or the development of any other cell type in the pancreas (Sander et al., 2000b). In Nkx6.1 mutant mice, a marked reduction in beta-cell numbers is first apparent at embryonic day (E)14, the time-point at which the first mature beta-cells differentiate. The specific defect in the beta-cell lineage and th...

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.

Gonadotropin-releasing hormone (GnRH) is the central regulator of reproductive function. Expression of the GnRH gene is confined to a rare population of neurons scattered throughout the hypothalamus. Restricted expression of the rat GnRH gene is driven by a multicomponent enhancer and an evolutionarily conserved promoter. Oct-1, a ubiquitous POU homeodomain transcription factor, was identified as an essential factor regulating GnRH transcription in the GT1-7 hypothalamic neuronal cell line. In this study, we conducted a two-hybrid interaction screen in yeast using a GT1-7 cDNA library to search for specific Oct-1 cofactors. Using this approach, we isolated Pbx1b, a TALE homeodomain transcription factor that specifically associates with Oct-1. We show that heterodimers containing Pbx/ Prep1 or Pbx/Meis1 TALE homeodomain proteins bind to four functional elements within the GnRH regulatory region, each in close proximity to an Oct-1-binding site. Cotransfection experiments indicate that TALE proteins are essential for GnRH promoter activity in the GT1-7 cells. Moreover, Pbx1 and Oct-1, as well as Prep1 and Oct-1, form functional complexes that enhance GnRH gene expression. Finally, Pbx1 is expressed in GnRH neurons in embryonic as well as mature mice, suggesting that the associations between TALE homeodomain proteins and Oct-1 regulate neuron-specific expression of the GnRH gene in vivo.

The GnRH gene is exclusively expressed in a discrete population of neurons in the hypothalamus. The promoter-proximal 173 bp of the rat GnRH gene are highly conserved through evolution and are bound by multiple nuclear proteins found in the neuronal cell line, GT1-7, a model for the GnRH-expressing hypothalamic neuron. To explore the protein-DNA interactions that occur within this promoter and the role of these interactions in targeting GnRH gene expression, we have mutagenized individual binding sites in this region. Deoxyribonuclease I protection experiments reveal that footprint 2, a 51-bp sequence that confers a 20-fold induction of the GnRH gene, is comprised of at least three independent protein-binding sites. Transfections of the GnRH promoter-reporter plasmid containing a series of block mutations of footprint 2 into GT1-7 neurons indicate that each of the three putative component sites contributes to transcriptional activity. Mutations in footprint 4 also decrease GnRH gene expression. Footprint 4 and the promoter-proximal site in footprint 2 contain octamer-like motifs, an element that is also present in the neuron-specific enhancer of the rat GnRH gene located approximately 1.6 kb upstream of the promoter. Previous studies in our laboratory have demonstrated that two enhancer octamer sites are bound by the POU-homeodomain transcription factor Oct-1 in GT1-7 cells. We now show that Oct-1 binds the octamer motifs within footprints 2 and 4. Thus, Oct-1 plays a critical role in the regulation of GnRH transcription, binding functional elements in both the distal enhancer and the promoter-proximal conserved region.

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GnRH gene expression is restricted to a tiny population of neurons scattered throughout the mediobasal hypothalamus. The combination of a 300-bp enhancer and the 173-bp promoter from the rat GnRH gene can confer this narrow specificity in transgenic mice and in transfections of hypothalamic GT1-7 cells. In the present study, we identify repeated CAATT elements in the 3' region of the rat GnRH enhancer that bind a tissue-restricted protein complex and play a significant role in cell-restricted expression of the GnRH gene. Deletions of multiple repeats demonstrate their importance in transcriptional activity. In fact, even mutation of a single repeat reduces expression. This reduction can be compensated by the conserved GnRH promoter, which also contains such elements and binds this protein complex. In Southwestern analysis, three proteins from GT1-7 nuclear extract bind to the CAATT element, and these proteins are not found in NIH3T3 cells. This cell-specific protein complex has properties of the Q50 homeodomain family of transcription factors and binds to as many as seven binding sites in the enhancer and promoter to play a key role in GnRH gene expression in the hypothalamus.