Glutamine synthetase is essential in early mouse embryogenesis

Abstract

Glutamine synthetase (GS) is expressed in a tissue-specific and developmentally controlled manner, and functions to remove ammonia or glutamate. Furthermore, it is the only enzyme that can synthesize glutamine de novo. Since congenital deficiency of GS has not been reported, we investigated its role in early development. Because GS is expressed in embryonic stem (ES) cells, we generated a null mutant by replacing one GS allele in-frame with a ␤-galactosidase-neomycine fusion gene. GS ؉/LacZ mice have no phenotype, but GS LacZ/LacZ mice die at ED3.5, demonstrating GS is essential in early embryogenesis. Although cells from ED2.5 GS LacZ/LacZ embryos and GS GFP/LacZ ES cells survive in vitro in glutaminecontaining medium, these GS-deficient cells show a reduced fitness in chimera analysis and fail to survive in tetraploid-complementation assays. The survival of heavily (>90%) chimeric mice up to at least ED16.5 indicates that GS deficiency does not entail cell-autonomous effects and that, after implantation, GS activity is not essential until at least the fetal period. We hypothesize that GS-deficient embryos die when they move from the uterine tube to the harsher uterine environment, where the embryo has to catabolize amino acids to generate energy and, hence, has to detoxify ammonia, which requires GS activity. Developmental Dynamics 236:1865-1875, 2007.

Astrocyte Reactivity and Reactive Astrogliosis: Costs and Benefits

“…Thus, whereas GS serves as an astrocyte marker, it does not appear as a good marker of reactive astrogliosis. The critical involvement of GS in normal brain development and function, demonstrated by an early mortality of both GS-deficient mice (92) and humans (87), points to the role of reduced GS expression and function in the pathogenesis of at least some CNS disorders. (194).…”

Glutamine Synthetase in Muscle Is Required for Glutamine Production during Fasting and Extrahepatic Ammonia Detoxification

“…Cryostat sections (10 m) were fixed in 0.5% buffered glutaraldehyde for 10 min at room temperature. ␤-Galactosidase activity was visualized by applying 5 mM K 3 [Fe(CN)] 6 , 5 mM K 4 [Fe(CN) 6 ], 10 mM EDTA (pH 8.0), 5 mM MgCl 2 , 20 mM NaCl, 0.1% (w/v) 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside, as described (18). For immunohistochemistry, the cryosections were briefly washed in phosphate-buffered saline to remove the optimal cutting temperature compound, postfixed in 4% buffered formaldehyde for 10 min at room temperature, washed again in phosphate-buffered saline, and boiled for 5 min in 10 mM sodium citrate (pH 6.0) to retrieve epitopes (21) and to inactivate endogenous alkaline phosphatase.…”

“…The sections were blocked in Teng-T (10 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 0.25% (w/v) gelatin, and 0.05% (v/v) Tween 20, pH 8.0) for 30 min and incubated overnight with the first antibody diluted in Teng-T (monoclonal anti-GS, 1:1,500 (Transduction Laboratories, Lexington, KY) and monoclonal anti-␤-galactosidase, 1: 500 (Promega Benelux, Leiden, The Netherlands)). After washing, the sections were incubated with a 1:100 dilution in Teng-T of goat anti-mouse IgG covalently coupled to alkaline phosphatase (Sigma), washed again, and incubated with alkaline-phosphatase substrate (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; Roche Applied Science) for 30 min (18). Incubations from which the first antibody was omitted served as negative controls.…”

Glutamine contributes to maintenance of mouse embryonic stem cell self-renewal through PKC-dependent downregulation of HDAC1 and DNMT1/3a

“…[4][5] Although Gln is another source of metabolic energy for cells beside glucose, [6][7] the physiological role of Gln in regulation of stem cell pluripotency is unclear. Gln synthetase, the only enzyme producing Gln de novo, is essential in early mouse embryogenesis due to its role in detoxification of ammonia production, 8 which suggests that Gln is essential in early embryogenesis and embryo development. 9 Furthermore, 2 unrelated human newborns with a congenital Gln synthetase deficiency presented with developmental disorders such as brain malformation, multiple organ failure, and infant death, [9][10] which prompted us to examine the effects of Gln on regulation of embryonic stem cell (ESC) function and pluripotency.…”