Transgenic expression of human equilibrative nucleoside transporter 1 in mouse neurons.

Transgenic mice that express human equilibrative nucleoside transporter subtype 1 (hENT1) under the control of a neuron-specific enolase promoter have been generated. Southern blot and PCR revealed the presence of the transgene in five founder mice. Mice from each founder line were examined by reverse transcriptase (RT)-PCR and found to express hENT1 in RNA isolated from whole brain, cerebral cortex, striatum, hippocampus, and cerebellum but not liver, kidney, heart, lung or skeletal muscle. Cortical synaptosomes prepared from transgenic mice had significantly increased [(3)H]adenosine uptake and [(3)H]nitrobenzylthioinosine binding, relative to samples from wild-type mice. In behavioral tests, transgenic mice had altered responses to caffeine and ethanol, two drugs that inhibit and enhance, respectively, adenosine receptor activity. Caffeine-induced locomotor stimulation was attenuated whereas the hypnotic effect of ethanol was enhanced in transgenic mice. Caffeine was more potent in inhibiting ethanol-induced motor incoordination in wild-type than in transgenic mice. No differences in expression of mouse genes for adenosine receptors, nucleoside transporters, or purine metabolizing enzymes were detected by RT-PCR analyses. These data indicate that expression of hENT1 in neurons does not trigger adaptive changes in expression of adenosine-related genes. Instead, hENT1 expression affects dynamic changes in endogenous adenosine levels, as revealed by altered behavioral responses to drugs that affect adenosine receptor signalling.

FGF-16 is required for embryonic heart development.

Fibroblast growth factor 16 (FGF-16) expression has previously been detected in mouse heart at mid-gestation in the endocardium and epicardium, suggesting a role in embryonic heart development. More specifically, exogenously applied FGF-16 has been shown to stimulate growth of embryonic myocardial cells in tissue explants. We have generated mice lacking FGF-16 by targeting the Fgf16 locus on the X chromosome. Elimination of Fgf16 expression resulted in embryonic death as early as day 11.5 (E11.5). External abnormalities, including hemorrhage in the heart and ventral body region as well as facial defects, began to appear in null embryos from E11.5. Morphological analysis of FGF-16 null hearts revealed cardiac defects including chamber dilation, thinning of the atrial and ventricular walls, and poor trabeculation, which were visible at E10.5 and more pronounced at E11.5. These findings indicate FGF-16 is required for embryonic heart development in mid-gestation through its positive effect on myocardial growth.

Proteomic identification of AP2 gamma as a rat placental lactogen II trophoblast cell-specific enhancer binding protein.

The factors that regulate the developmental expression of the rodent prolactin gene family in placenta remain poorly defined. We previously identified an enhancer element in the 5' flanking region of one family member, rat placental lactogen II (rPLII), which could target reporter gene expression to the placenta in transgenic mice; this enhancer functioned in the Rcho rat trophoblast cell line but not in the rat pituitary GC cell line. In further experiments to identify the factors that bind this element, we have selectively enriched for DNA binding proteins in nuclear extract from Rcho cells using magnetic beads coupled to a 43-bp enhancer oligonucleotide. Tryptic peptides of bound proteins were analyzed by HPLC coupled off-line to matrix-assisted laser desorption ionization time of flight mass spectrometry. Several peptides of AP2 gamma, a key trophoblast cell-specific transcription factor, were identified. Gel mobility shift assays using AP2 gamma-specific antiserum and mutant enhancer oligonucleotides demonstrated binding specifically to the FP2 DNase I-protected region of the element, identifying an atypical binding site for this factor. In cotransfection assays in rat pituitary GC cells, AP2 gamma transactivated the enhancer via this region. Chromatin immunoprecipitation assays confirmed AP2 gamma occupancy of the enhancer region in situ in the nuclei of Rcho giant cells. These data support a role for AP2 gamma in the placental giant cell-specific expression of the rPLII gene and provide the first direct evidence for the involvement of a placental-specific transcription factor in the regulation of a member of this gene family.

Defining regulatory regions in the rat prolactin gene family locus using a large P1 genomic clone.

Members of the large rat prolactin gene family located on chromosome 17 are expressed in one or more placental trophoblast cell types and maternal decidua at specific times during pregnancy. Studies to identify the factors involved in these highly specific developmental expression patterns, using limited amounts of 5'-flanking DNA, have met with only partial success. Here we report the isolation and characterization of an 80-kb rat genomic clone, P1 12830, containing linked rat placental lactogen II, rat prolactin-like protein-I, and rat prolactin-like protein-B genes with substantial amounts of 5'- and 3'-flanking DNA as well as a rat placental lactogen II-related pseudogene, the first to be described in this gene family. This clone was used to create F0 transgenic mice, and the levels of expression of the three rat genes were compared with those of the endogenous mouse genes, using RT-PCR. Each rat gene was expressed differently in the same placenta, confirming the importance of sufficient flanking sequences in the expression of the individual genes. These studies emphasize the need for large genomic clones in defining the complete complement of factors that regulate the developmental expression of the rat prolactin gene locus.

Embryonic survival and severity of cardiac and craniofacial defects are affected by genetic background in fibroblast growth factor-16 null mice.

Disruption of the X-chromosome fibroblast growth factor 16 (Fgf-16) gene, a member of the FGF-9 subfamily with FGF-20, was linked with an effect on cardiac development in two independent studies. However, poor trabeculation with lethality by embryonic day (E) 11.5 was associated with only one, involving maintenance in Black Swiss (Bsw) versus C57BL/6 mice. The aim of this study was to examine the potential influence of genetic background through breeding the null mutation onto an alternate (C57BL/6) background. After three generations, 25% of Fgf-16(-/Y) mice survived to adulthood, which could be reversed by reducing the contribution of the C57BL/6 genetic background by back crossing to another strain. There was no significant difference between FGF-9 and FGF-20 RNA levels in Fgf-16 null versus wild-type mice regardless of strain. However, FGF-8 RNA levels were reduced significantly in Bsw but not C57BL/6 mice. FGF-8 is linked to anterior heart development and like the FGF-9 subfamily is reportedly expressed at E10.5. Like FGF-16, neuregulin as well as signaling via ErbB2 and ErbB4 receptors have been linked to trabeculae formation and cardiac development around E10.5. Basal neuregulin, ErbB2, and ErbB4 as well as FGF-8, FGF-9, and FGF-16 RNA levels varied in Bsw versus C57BL/6 mice. These data are consistent with the ability of genetic background to modify the phenotype and affect embryonic survival in Fgf-16 null mice.