Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines.

Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation. Using morula aggregation to produce chimeras, five newly derived 129/SvEv and two C57BL/6 ES cell lines tested at early passages were found to contribute extensively to chimeras and produce germline-transmitting male chimeras. Furthermore, the two 129S/vEv ES cell lines that were tested and one of the C57BL/6 ES cell lines were able to maintain these characteristics after many passages in vitro. Our results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages. C56BL/6-derived ES cell lines, however, have a greater tendency than 129-derived ES cell lines to lose their ability to colonize the germline.

Increased LDL cholesterol and atherosclerosis in LDL receptor-deficient mice with attenuated expression of scavenger receptor B1.

Scavenger receptor BI (SR-BI) is a multiligand cell-surface receptor that plays a central role in high density lipoprotein homeostasis in rodents. To investigate a role for SR-BI in atherosclerosis, mice with attenuated SR-BI expression were crossed with low density lipoprotein (LDL) receptor-deficient mice. Compound-homozygous mutants showed increased plasma cholesterol, surprisingly due primarily to increased LDL cholesterol and apolipoprotein B levels. LDL turnover studies showed that this resulted from increased LDL cholesterol production rather than decreased LDL catabolism. Atherosclerotic lesion size was significantly increased in male compound-mutant mice relative to LDL receptor-deficient controls (93 427+/-16 079 versus 34 448+/-5 331 microm(2), respectively; P=0.003). The proatherogenic effect of attenuated SR-BI expression may in part be due to increased LDL cholesterol levels. These findings suggest that upregulation of the receptor could have therapeutic potential for the treatment of atherosclerosis.

A role for smad6 in development and homeostasis of the cardiovascular system.

Smad proteins are intracellular mediators of signalling initiated by Tgf-betasuperfamily ligands (Tgf-betas, activins and bone morphogenetic proteins (Bmps)). Smads 1, 2, 3, 5 and 8 are activated upon phosphorylation by specific type I receptors, and associate with the common partner Smad4 to trigger transcriptional responses. The inhibitory Smads (6 and 7) are transcriptionally induced in cultured cells treated with Tgf-beta superfamily ligands, and downregulate signalling in in vitro assays. Gene disruption in mice has begun to reveal specific developmental and physiological functions of the signal-transducing Smads. Here we explore the role of an inhibitory Smad in vivo by targeted mutation of Madh6 (which encodes the Smad6 protein). Targeted insertion of a LacZ reporter demonstrated that Smad6 expression is largely restricted to the heart and blood vessels, and that Madh6 mutants have multiple cardiovascular abnormalities. Hyperplasia of the cardiac valves and outflow tract septation defects indicate a function for Smad6 in the regulation of endocardial cushion transformation. The role of Smad6 in the homeostasis of the adult cardiovascular system is indicated by the development of aortic ossification and elevated blood pressure in viable mutants. These defects highlight the importance of Smad6 in the tissue-specific modulation of Tgf-beta superfamily signalling pathways in vivo.

Targeted deletion of the tub mouse obesity gene reveals that tubby is a loss-of-function mutation.

The mouse tubby phenotype is characterized by maturity-onset obesity accompanied by retinal and cochlear degeneration. A positional cloning effort to find the gene responsible for this phenotype led to the identification of tub, a member of a novel gene family of unknown function. A splice defect mutation in the 3' end of the tub gene, predicted to disrupt the C terminus of the Tub protein, has been implicated in the genesis of the tubby phenotype. It is not clear, however, whether the Tub mutant protein retains any biological activity, or perhaps has some dominant function, nor is it established that the tubby mutation is itself responsible for all of the observed tubby phenotypes. To address these questions, we generated tub-deficient mice and compared their phenotype to that of tubby mice. Our results demonstrate that tubby is a loss-of-function mutation of the tub gene and that loss of the tub gene is sufficient to give rise to the full spectrum of tubby phenotypes. We also demonstrate that loss of photoreceptors in the retina of tubby and tub-deficient mice occurs by apoptosis. In addition, we show that Tub protein expression is not significantly altered in the ob, db, or melanocortin 4 receptor-deficient mouse model of obesity.

Decreased selective uptake of high density lipoprotein cholesteryl esters in apolipoprotein E knock-out mice.

Scavenger receptor BI (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters (CE) by cells, i.e., the uptake of CE without degradation of HDL protein. Mice with attenuated expression of SR-BI, because of targeted gene mutation (SR-BIatt mice), have increased plasma HDL levels as a result of decreased selective uptake in the liver. To further evaluate the role of SR-BI in lipoprotein metabolism, compound apolipoprotein E knock-out (apoE0)/SR-BIatt mice were bred. Hepatic SR-BI protein was increased (2.3-fold) in apoE0 mice compared with wild type (wt) and was reduced significantly in apoE0/SR-BIatt mice. However, the plasma lipoprotein profile of apoE0 and apoE0/SR-BIatt mice was identical. This was explained by HDL turnover studies that revealed that the selective clearance of HDL CE by the liver and adrenal was already profoundly impaired in apoE0 mice compared with wt (28% of wt in liver). A similar decrease in selective uptake was seen when apoE0 HDL was incubated with isolated apoE0 hepatocytes. The results suggest that apoE plays a major role in the selective clearance of HDL CE by the liver and adrenal gland, possibly by facilitating the presentation of HDL to SR-BI at the cell surface.

The mahogany protein is a receptor involved in suppression of obesity.

Genetic studies have shown that mutations within the mahogany locus suppress the pleiotropic phenotypes, including obesity, of the agouti-lethal-yellow mutant. Here we identify the mahogany gene and its product; this study, to our knowledge, represents the first positional cloning of a suppressor gene in the mouse. Expression of the mahogany gene is broad; however, in situ hybridization analysis emphasizes the importance of its expression in the ventromedial hypothalamic nucleus, a region that is intimately involved in the regulation of body weight and feeding. We present new genetic studies that indicate that the mahogany locus does not suppress the obese phenotype of the melanocortin-4-receptor null allele or those of the monogenic obese models (Lep(db), tub and Cpe(fat)). However, mahogany can suppress diet-induced obesity, the mechanism of which is likely to have implications for therapeutic intervention in common human obesity. The amino-acid sequence of the mahogany protein suggests that it is a large, single-transmembrane-domain receptor-like molecule, with a short cytoplasmic tail containing a site that is conserved between Caenorhabditis elegans and mammals. We propose two potential, alternative modes of action for mahogany: one draws parallels with the mechanism of action of low-affinity proteoglycan receptors such as fibroblast growth factor and transforming growth factor-beta, and the other suggests that mahogany itself is a signalling receptor.

Targeted mutation reveals a central role for SR-BI in hepatic selective uptake of high density lipoprotein cholesterol.

Scavenger receptor BI (SR-BI) is a cell surface receptor that binds high density lipoproteins (HDL) and mediates selective uptake of HDL cholesteryl esters (CE) in transfected cells. To address the physiological role of SR-BI in HDL cholesterol homeostasis, mice were generated bearing an SR-BI promoter mutation that resulted in decreased expression of the receptor in homozygous mutant (designated SR-BI att) mice. Hepatic expression of the receptor was reduced by 53% with a corresponding increase in total plasma cholesterol levels of 50-70% in SR-BI att mice, attributable almost exclusively to elevated plasma HDL. In addition to increased HDL-CE, HDL phospholipids and apo A-1 levels were elevated, and there was an increase in HDL particle size in mutant mice. Metabolic studies using HDL bearing nondegradable radiolabels in both the protein and lipid components demonstrated that reducing hepatic SR-BI expression by half was associated with a decrease of 47% in selective uptake of CE by the liver, and a corresponding reduction of 53% in selective removal of HDL-CE from plasma. Taken together, these findings strongly support a pivotal role for hepatic SR-BI expression in regulating plasma HDL levels and indicate that SR-BI is the major molecule mediating selective CE uptake by the liver. The inverse correlation between plasma HDL levels and atherosclerosis further suggests that SR-BI may influence the development of coronary artery disease.

Targeted disruption of the melanocortin-4 receptor results in obesity in mice.

The melanocortin-4 receptor (MC4-R) is a G protein-coupled, seven-transmembrane receptor expressed in the brain. Inactivation of this receptor by gene targeting results in mice that develop a maturity onset obesity syndrome associated with hyperphagia, hyperinsulinemia, and hyperglycemia. This syndrome recapitulates several of the characteristic features of the agouti obesity syndrome, which results from ectopic expression of agouti protein, a pigmentation factor normally expressed in the skin. Our data identify a novel signaling pathway in the mouse for body weight regulation and support a model in which the primary mechanism by which agouti induces obesity is chronic antagonism of the MC4-R.

Gonadotrope-specific expression of the human follicle-stimulating hormone beta-subunit gene in pituitaries of transgenic mice.

The molecular basis for the cell-specific and hormonal regulation of the pituitary gonadotropin beta-subunit genes is unknown. To identify the essential DNA regulatory elements of the human FSH beta (hFSH beta) gene, we introduced a 10-kilobase cloned fragment encompassing the gene into the germline of transgenic mice. We showed by a combination of Northern blot hybridization, RIA, and immunofluorescence histochemistry that the hFSH beta gene was expressed exclusively in mouse pituitary gonadotropes. Human FSH beta was actively secreted, presumably as an interspecies heterodimer, with mouse alpha-subunit, and no free hFSH beta was detected in serum. Male transgenic mice consistently had higher basal pituitary expression and secretion of hFSH beta than the female transgenic mice. Castration combined with testosterone replacement caused a marked reduction in hFSH beta gene expression in transgenic males, but had no effect on the pituitary levels of FSH in normal male mice. These experiments demonstrate that the essential DNA regulatory elements and gonadotrope transcription factors required for cell-specific expression of the FSH beta gene must be conserved between human and mouse. There appear to be important differences in the hormonal regulation of the gene between species, however.

Pituitary-specific and hormonally regulated gene expression directed by the rat proopiomelanocortin promoter in transgenic mice.

All aspects of POMC biosynthesis exhibit tissue-specific regulation. The single copy gene is highly expressed in anterior lobe (AL) corticotrophs and intermediate lobe (IL) melanotrophs of the pituitary gland and in the arcuate nucleus of the hypothalamus. POMC gene transcription in corticotrophs is induced by hypothalamic CRH and vasopressin and inhibited by adrenal glucocorticoids, while in melanotrophs it is predominantly regulated by beta-adrenergic neural input and dopamine. To identify the rat POMC (rPOMC) gene sequences necessary and sufficient to target expression and hormonal regulation in corticotrophs and melanotrophs, we generated 13 transgenic mice carrying rPOMC fusion genes. The genes consisted of 706 or 480 basepairs of rPOMC 5' flanking sequences ligated to either the E. coli LacZ gene encoding beta-galactosidase or the K1 mutant of the SV40 large T-antigen gene. Overall, half of the transgenic lines had reporter gene expression in their AL and IL in a pattern indistinguishable from ACTH immunohistochemistry. In three of these lines, beta-galactosidase or K1 T-antigen was localized by double immunofluorescence exclusively to ACTH-positive corticotrophs and melanotrophs. Transcriptional regulation of the rPOMC-LacZ fusion gene in response to hormonal manipulation was quantified by a fluorescence assay for beta-galactosidase enzyme activity in pituitary extracts. There was a 15-fold increase in AL enzyme activity after adrenalectomy and a 3-fold increase in IL activity after haloperidol treatment. X-gal histochemistry of pituitaries from hormonally treated mice confirmed the cellular specificity of these effects.(ABSTRACT TRUNCATED AT 250 WORDS)