The alpha1-fetoprotein (AFP) gene is located between the albumin and alpha-albumin genes and is activated by transcription factor FTF (fetoprotein transcription factor), presumed to transduce early developmental signals to the albumin gene cluster. We have identified FTF as an orphan nuclear receptor of the Drosophila FTZ-F1 family. FTF recognizes the DNA sequence 5'-TCAAGGTCA-3', the canonical recognition motif for FTZ-F1 receptors. cDNA sequence homologies indicate that rat FTF is the ortholog of mouse LRH-1 and Xenopus xFF1rA. Rodent FTF is encoded by a single-copy gene, related to the gene encoding steroidogenic factor 1 (SF-1). The 5.2-kb FTF transcript is translated from several in-frame initiator codons into FTF isoforms (54 to 64 kDa) which appear to bind DNA as monomers, with no need for a specific ligand, similar KdS (approximately equal 3 x 10(-10) M), and similar transcriptional effects. FTF activates the AFP promoter without the use of an amino-terminal activation domain; carboxy-terminus-truncated FTF exerts strong dominant negative effects. In the AFP promoter, FTF recruits an accessory trans-activator which imparts glucocorticoid reactivity upon the AFP gene. FTF binding sites are found in the promoters of other liver-expressed genes, some encoding liver transcription factors; FTF, liver alpha1-antitrypsin promoter factor LFB2, and HNF-3beta promoter factor UF2-H3beta are probably the same factor. FTF is also abundantly expressed in the pancreas and may exert differentiation functions in endodermal sublineages, similar to SF-1 in steroidogenic tissues. HepG2 hepatoma cells seem to express a mutated form of FTF.

Development of the mammalian embryo relies upon nutritive functions fulfilled by the visceral endoderm and then by the liver (1). A part of these functions is accomplished by nutrient carrier proteins of the albumin gene family, a multigene locus expressed by the liver and subject to precise developmental controls. One albumin-related gene, the ␣ 1 -fetoprotein (AFP) 1 gene, is activated at the onset of liver differentiation and operates tightly coupled with liver growth (2, 3). In 1988, our group circumscribed a proximal AFP promoter element essential to AFP gene activity in hepatocytes, and distinct from promoter components regulating the other albumin loci (4). The AFP-specific activator was then identified as orphan receptor fetoprotein transcription factor (5-7), so named for its first identified target locus (genome data base nomenclature, 2 NR5A2 in the nuclear receptor nomenclature, Ref. 9); also referred to as LRH1 or CPF). FTF belonged to a primitive class of nuclear receptors and emerged as a critical lead to connect AFP gene activation with early embryonic growth and differentiation processes.Subsequent studies indicated that developmental FTF functions even preceded its activation of the AFP locus in hepatocytes. In situ hybridization analysis in the mouse at embryonic day 8 -9 showed abundant FTF transcripts in the foregut endoderm, before liver morphogenesis (10). Characterization of the FTF gene promoter also revealed a cluster of regulatory motifs conserved in distant species and potential targets of cell lineage specification factors (11). Among these were three proximal binding sites for GATA factors, known to be essential for visceral endoderm function (12, 13). Furthermore, three HNF genes important to liver differentiation, HNF1␣, HNF4␣, and HNF3␤, were found to each contain double FTF-binding sites in their proximal promoter and to be activated by FTF in transfection assays (11). Thus, a pivotal role was suggested for FTF in a transcriptional cascade using determination factors to activate FTF in prehepatic endodermal cells, and then using FTF to drive AFP and other effectors of the hepatic program.

Steroidogenic factor-1 (SF-1) (NR5A1) is an orphan nuclear receptor that plays a premier role in ovarian organogenesis. Recent studies document mRNA expression of the structurally related factor NR5A2 (FTF, LRH-1, SF-2) in the adult ovary and more specifically in granulosa cells and luteal cells but not theca cells. Conversely, SF-1 was shown to be expressed at higher levels in theca/interstitial cells. These latter observations raised the possibility that FTF/LRH-1 may control target gene expression in granulosa cells of developing follicles. Using quantitative PCR our results show that FTF/LRH-1 message is expressed at higher levels in the ovary than in liver or other tissues analyzed. We show by in situ hybridization and LacZ expression in ovaries of transgenic mice bearing an FTF-promoter-LacZ fusion gene that FTF/LRH-1 is selectively expressed in granulosa cells of rat and mouse ovaries and is not present in theca cells or interstitial cells. However, by a variety of approaches, we showed that SF-1 mRNA and protein are expressed in greater amounts than FTF/LRH-1 in granulosa cells of follicles at all stages of development. Expression of SF-1 mRNA and protein in granulosa cells was verified by in situ hybridization, immunohistochemistry of ovarian sections, and immunocytochemistry of cultured rat granulosa cells. The significance of SF-1 in regulating target gene activation was supported by EMSA. An abundant granulosa cell protein binding to the SF-1-binding motif (CCAAGGTCA) present in the aromatase promoter and an FTF/LRH-1 motif (TGTCCTTGAACA) in the alpha-fetoprotein promoter was supershifted by two SF-1-specific antibodies but not by an FTF antibody. Conversely, with the same probes, a less abundant protein/DNA complex present in liver and ovarian cell extracts was shifted by an FTF antibody but not by the SF-1 antibodies. SF-1 and FTF/LRH-1 were differentially regulated in vivo by estradiol, FSH and prolactin. Collectively these data indicate that granulosa cells of small and preovulatory follicles express both SF-1 and FTF/LRH-1 and that each orphan receptor may regulate target gene expression in these cells.

The North American greater snow goose population has increased dramatically during the last 40 years. We evaluated whether refuge creation, changes in land use on the wintering and staging grounds, and climate warming have contributed to this expansion by affecting the distribution, habitat use, body condition, and migration phenology of birds. We also reviewed the effects of the increasing population on marshes on the wintering grounds, along the migratory routes and on the tundra in summer. Refuges established before 1970 may have contributed to the initial demographic increase. The most important change, however, was the switch from a diet entirely based on marsh plants in spring and winter (rhizomes of Scirpus/Spartina) to one dominated by crops (corn/young grass shoots) during the 1970s and 1980s. Geese now winter further north along the US Atlantic coast, leading to reduced hunting mortality. Their migratory routes now include portions of southwestern Québec where corn production has increased exponentially. Since the mid-1960s, average temperatures have increased by 1-2.4 1C throughout the geographic range of geese, which may have contributed to the northward shift in wintering range and an earlier migration in spring. Access to spilled corn in spring improved fat reserves upon departure for the Arctic and may have contributed to a high fecundity. The population increase has led to intense grazing of natural wetlands used by geese although these habitats are still largely undamaged. The foraging in fields allowed the population to exceed limits imposed by natural marshes in winter and spring, but also prevented permanent damage because of their overgrazing.

Fetoprotein transcription factor (FTF) is an orphan nuclear receptor that activates the ␣ 1 -fetoprotein gene during early liver developmental growth. Here we sought to define better the position of FTF in transcriptional cascades leading to hepatic differentiation. The mouse FTF gene was isolated and assigned to chromosome 1 band E4 (one mFTF pseudogene was also found). Exon/intron mapping shows an mFTF gene structure similar to that of its close homologue SF1, with two more N-terminal exons in the mFTF gene; exon mapping also delimits several FTF mRNA 5-and 3-splice variants. The mFTF transcription initiation site was located in adult liver at 238 nucleotides from the first translation initiator codon, with six canonical GATA, E box, and Nkx motifs clustered between ؊50/؊140 base pairs (bp) from the cap site; DNA/protein binding assays also pinpointed an HNF4-binding element at ؉36 bp and an FTFbinding element at ؊257 bp. Transfection assays and point mutations showed that the mFTF promoter is activated by GATA, HNF4␣, FTF, Nkx, and basic helixloop-helix factors, with marked cooperativity between GATA and HNF4␣. A tandem GATA/E box activatory motif in the proximal mFTF promoter is strikingly similar to a composite motif coactivated by differentiation inducers in the hematopoietic lineage; a tandem GATANkx motif in the distal mFTF promoter is also similar to a composite motif transducing differentiation signals from transforming growth factor-␤-like receptors in the cardiogenic lineage. Three genes encoding transcription factors critical to early hepatic differentiation, Hnf3␤, Hnf4␣, and Hnf1␣, each contain dual FTF-binding elements in their proximal promoters, and all three promoters are activated by FTF in transfection assays. Direct DNA binding action and cooperativity was demonstrated between FTF and HNF3␤ on the Hnf3␤ promoter and between FTF and HNF4␣ on the Hnf1␣ promoter. These combined results suggest that FTF is an early intermediary between endodermal specification signals and downstream genes that establish and amplify the hepatic phenotype.At 8 -8.5 days of mouse embryogenesis, endodermal cells of the ventral foregut interact with the cardiac mesoderm and become committed to the hepatic differentiation program; these newly specified cells then migrate and proliferate in the mesenchyme of the septum transversum where liver morphogenesis becomes apparent at ϷE10.5 (1, 2). Initial induction of hepatic functions is driven in part by growth factors of the FGF 1 family, secreted by cardiac mesodermal cells and acting via transmembrane receptor kinases at the endodermal cell surface (3). The process also involves potentiating transcription factors among which GATA factors appear essential to endodermal determination across vertebrates as well as invertebrates (Ref. 4 and references therein). Following liver specification, transcriptional activation cascades further develop among early hepatic transcription factors creating interactive regulatory networks that amplify the induction signals and imprint the...

Mutations were introduced in 7 kilobases of 5'-flanking rat ac-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.The a,-fetoprotein (AFP) gene, a member of the albumin gene family, is expressed by fetal or malignant hepatocytes and repressed in normal mature hepatocytes. This forms the basis of a long-exploited model system to study cell differentiation and its impairment in neoplasia (1). Molecular genetics have brought the AFP model to molecular levels of cell differentiation, toward finely discerning how genes respond to or escape from developmental and growth signals (2,40,41). As yet, little is known about how differential regulation is exerted on the tandemly organized AFPalbumin locus and whether the two genes operate under shared elements of control. However, at least one wellcharacterized transcription factor, the glucocorticoid receptor, is known to selectively shut off the AFP gene in the developing liver (4, 21). This hormonal effect reaches into the mechanisms of neoplastic resistance to differentiation, because the AFP gene in malignant cells is generally refractory to glucocorticoids (2).Analyses of rat AFP gene and chromatin structures have identified domains in the 5' region of the locus potentially involved in its liver-specific, developmental stage-dependent, and glucocorticoid-regulated expression. A promoter domain spanning '230 nucleotides comprises a chromatin DNase I-hypersensitive (DH) site (32, 42, 44) that is selectively suppressed by dexamethasone (44), glucocorticoid receptor recognition sequences, and octamer motifs similar to those present in other genes under developmental and growth control (8). A distal domain (-2 to -4 kilobases [kb] relative to the AFP trans...

During organogenesis, the winged helix hepatocyte nuclear factor 3beta (HNF-3beta) protein participates in regulating gene transcription in the developing esophagus, trachea, liver, lung, pancreas, and intestine. Hepatoma cell transfection studies identified a critical HNF-3beta promoter factor, named UF2-H3beta, and here, we demonstrate that UF2-H3beta is identical to the fetoprotein transcription factor (FTF). In situ hybridization studies of mouse embryos demonstrate that FTF expression initiates in the foregut endoderm during liver and pancreatic morphogenesis (day 9) and that earlier expression of FTF is observed in the yolk sac endoderm, branchial arch and neural crest cells (day 8). Abundant FTF hybridization signals are observed throughout morphogenesis of the liver, pancreas, and intestine and its expression continues in the epithelial cells of these adult organs. In day 17 mouse embryos and adult pancreas, however, expression of FTF becomes restricted to the exocrine acinar and ductal epithelial cells.

No abstract