Abnormal serotonergic development in a mouse model for the Smith-Lemli-Opitz syndrome: implications for autism.

The Smith-Lemli-Opitz syndrome (SLOS) is a malformation/mental retardation syndrome resulting from an inborn error in 3beta-hydroxysteroid Delta7-reductase (DHCR7), the terminal enzyme required for cholesterol biosynthesis. Using a targeting strategy designed to virtually eliminate Dhcr7 activity, we have created a SLOS mouse model that exhibits commissural deficiencies, hippocampal abnormalities, and hypermorphic development of serotonin (5-HT) neurons. The latter is of particular interest with respect to current evidence that serotonin plays a significant role in autism spectrum disorders and the recent clinical observation that 50% of SLOS patients present with autistic behavior. Immunohistochemical analyses have revealed a 306% increase in the area of 5-HT immunoreactivity (5-HT IR) in the hindbrains of mutant (Dhcr7-/-) mice as compared to age-matched wild type animals. Amount of 5-HT IR was measured as total area of IR per histological section. Additionally, a regional increase as high as 15-fold was observed for the most lateral sagittal hindbrain sections. In Dhcr7-/- mice, an expansion of 5-HT IR into the ventricular zone and floor plate region was observed. In addition, the rostral and caudal raphe groups exhibited a radial expansion in Dhcr7-/- mice, with 5-HT IR cells present in locations not seen in wild type mice. This increase in 5-HT IR appears to represent an increase in total number of 5-HT neurons and fibers. These observations may help explain the behavioral phenotype seen in SLOS, and provide clues for future therapeutic interventions that utilize pharmacological modulation of the serotonergic system.

7-Dehydrocholesterol-dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome.

Smith-Lemli-Opitz/RSH syndrome (SLOS), a relatively common birth-defect mental-retardation syndrome, is caused by mutations in DHCR7, whose product catalyzes an obligate step in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. A null mutation in the murine Dhcr7 causes an identical biochemical defect to that seen in SLOS, including markedly reduced tissue cholesterol and total sterol levels, and 30- to 40-fold elevated concentrations of 7-dehydrocholesterol. Prenatal lethality was not noted, but newborn homozygotes breathed with difficulty, did not suckle, and died soon after birth with immature lungs, enlarged bladders, and, frequently, cleft palates. Despite reduced sterol concentrations in Dhcr7(-/-) mice, mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for sterol biosynthesis, the LDL receptor, and SREBP-2 appeared neither elevated nor repressed. In contrast to mRNA, protein levels and activities of HMG-CoA reductase were markedly reduced. Consistent with this finding, 7-dehydrocholesterol accelerates proteolysis of HMG-CoA reductase while sparing other key proteins. These results demonstrate that in mice without Dhcr7 activity, accumulated 7-dehydrocholesterol suppresses sterol biosynthesis posttranslationally. This effect might exacerbate abnormal development in SLOS by increasing the fetal cholesterol deficiency.

Synthesis of a mouse model of the dysfibrinogen Vlissingen/Frankfurt IV.

The dysfibrinogen Vlissingen/Frankfurt IV is characterized as a deletion of Asn319 and Asp320 from the C-terminus of the gamma-chain of fibrinogen. This dysfibrinogen, which was identified in several family members that are all heterozygous for the in-frame 6-bp deletion, is associated with both venous and arterial thrombosis. Here, we describe the generation of a murine model of the V/F IV dysfibrinogen using gene targeting of mouse gamma-chain DNA. Preliminary analysis shows that the human and mouse variant fibrinogens are similar: analogous to the human V/F IV protein, the D1 fragment of the variant mouse fibrinogen is partially protected from digestion in the presence of calcium or Gly-Pro-Arg-Pro. These heterozygous mice provide the first opportunity to examine the association of thrombophilia and dysfibrinogenemia in a controlled genetic background.

Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II.

The classically recognized functions of the renin-angiotensin system are mediated by type 1 (AT1) angiotensin receptors. Whereas man possesses a single AT1 receptor, there are two AT1 receptor isoforms in rodents (AT1A and AT1B) that are products of separate genes (Agtr1a and Agtr1b). We have generated mice lacking AT1B (Agtr1b -/-) and both AT1A and AT1B receptors (Agtr1a -/-Agtr1b -/-). Agtr1b -/- mice are healthy, without an abnormal phenotype. In contrast, Agtr1a -/-Agtr1b -/- mice have diminished growth, vascular thickening within the kidney, and atrophy of the inner renal medulla. This phenotype is virtually identical to that seen in angiotensinogen-deficient (Agt-/-) and angiotensin-converting enzyme-deficient (Ace -/-) mice that are unable to synthesize angiotensin II. Agtr1a -/-Agtr1b -/- mice have no systemic pressor response to infusions of angiotensin II, but they respond normally to another vasoconstrictor, epinephrine. Blood pressure is reduced substantially in the Agtr1a -/- Agtr1b -/- mice and following administration of an angiotensin converting enzyme inhibitor, their blood pressure increases paradoxically. We suggest that this is a result of interruption of AT2-receptor signaling. In summary, our studies suggest that both AT1 receptors promote somatic growth and maintenance of normal kidney structure. The absence of either of the AT1 receptor isoforms alone can be compensated in varying degrees by the other isoform. These studies reaffirm and extend the importance of AT1 receptors to mediate physiological functions of the renin-angiotensin system.

Targeted disruption of the mouse topoisomerase I gene by camptothecin selection.

Topoisomerase I has ubiquitous roles in important cellular functions such as replication, transcription, and recombination. In order to further characterize this enzyme in vivo, we have used gene targeting to inactivate the mouse Top-1 gene. A selection protocol using the topoisomerase I inhibitor camptothecin facilitated isolation of embryonic stem cell clones containing an inactivated allele; isolation of correctly targeted clones was enhanced 75-fold over that achieved by normal selection procedures. The disrupted Top-1 allele is embryonic lethal when homozygous, and development of such embryos fails between the 4- and 16-cell stages. Both sperm and oocytes containing the inactive allele maintain viability through the fertilization point, and thus gene expression of topoisomerase I is not required for gamete viability. These studies demonstrate that topoisomerase I is essential for cell growth and division in vivo. The Top-1 gene was also shown to be linked to the agouti locus.

Hepatocellular and hepatic peroxisomal alterations in mice with a disrupted peroxisomal fatty acyl-coenzyme A oxidase gene.

Peroxisomal genetic disorders, such as Zellweger syndrome, are characterized by defects in one or more enzymes involved in the peroxisomal beta-oxidation of very long chain fatty acids and are associated with defective peroxisomal biogenesis. The biologic role of peroxisomal beta-oxidation system, which consists of three enzymes: fatty acyl-CoA oxidase (ACOX), enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), and thiolase, has been examined in mice by disrupting ACOX gene, which encodes the first and rate-limiting enzyme of this system. Homozygous (ACOX -/-) mice lacked the expression of ACOX protein and accumulate very long chain fatty acids in blood. However, these homozygous mice are viable, but growth-retarded and infertile. During the first 3-4 months of age, the livers of ACOX -/- mice reveal severe microvesicular fatty metamorphosis of hepatocytes. In such steatotic cells, peroxisome assembly is markedly defective; as a result, they contain few or no peroxisomes. Few hepatocytes in 1-3-month-old ACOX -/- mice contain numerous peroxisomes, and these peroxisome-rich hepatocytes show no fatty change. At this stage, the basal mRNA levels of HD, thiolase, and other peroxisome proliferator-induced target genes were elevated in ACOX -/- mouse liver, but these mice, when treated with a peroxisome proliferator, showed no increases in the number of hepatic peroxisomes and in the mRNAs levels of these target genes. Between 4 and 5 months of age, severe steatosis resulted in scattered cell death, steatohepatitis, formation of lipogranulomas, and focal hepatocellular regeneration. In 6-7-month-old animals, the newly emerging hepatocytes, which progressively replaced steatotic cells, revealed spontaneous peroxisome proliferation. These livers showed marked increases in the mRNA levels of the remaining two genes of the beta-oxidation system, suggesting that ACOX gene disruption leads to increased endogenous ligand-mediated transcription levels. These observations demonstrate links among peroxisomal beta-oxidation, development of severe microvesicular fatty liver, peroxisome assembly, cell death, and cell proliferation in liver.

Single-copy transgenic mice with chosen-site integration.

We describe a general way of introducing transgenes into the mouse germ line for comparing different sequences without the complications of variation in copy number and insertion site. The method uses homologous recombination in embryonic stem (ES) cells to generate mice having a single copy of a transgene integrated into a chosen location in the genome. To test the method, a single copy murine bcl-2 cDNA driven by either a chicken beta-actin promoter or a human beta-actin promoter has been inserted immediately 5' to the X-linked hypoxanthine phosphoribosyltransferase locus by a directly selectable homologous recombination event. The level of expression of the targeted bcl-2 transgene in ES cells is identical in independently isolated homologous recombinants having the same promoter yet varies between the different promoters. In contrast, the expression of bcl-2 transgenes having the same (chicken beta-actin) promoter varies drastically when they are independently integrated at random insertion sites. Both promoters direct broad expression of the single-copy transgene in mice derived from the respective targeted ES cells. In vitro and in vivo, the human beta-actin promoter consistently directed a higher level of transgene expression than the chicken beta-actin promoter.

Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse.

The prostaglandin endoperoxide H synthase isoform 2, cyclooxygenase 2 (COX-2), is induced at high levels in migratory and other responding cells by pro-inflammatory stimuli. COX-2 is generally considered to be a mediator of inflammation. Its isoform, COX-1, is constitutively expressed in most tissues and is thought to mediate "housekeeping" functions. These two enzymes are therapeutic targets of the widely used nonsteroidal anti-inflammatory drugs (NSAIDs). To investigate further the different physiologic roles of these isoforms, we have used homologous recombination to disrupt the mouse gene encoding COX-2 (Ptgs2). Mice lacking COX-2 have normal inflammatory responses to treatments with tetradecanoyl phorbol acetate or with arachidonic acid. However, they develop severe nephropathy and are susceptible to peritonitis.

Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration.

Cyclooxygenases 1 and 2 (COX-1 and COX-2) are key enzymes in prostaglandin biosynthesis and the target enzymes for the widely used nonsteroidal anti-inflammatory drugs. To study the physiological roles of the individual isoforms, we have disrupted the mouse Ptgs1 gene encoding COX-1. Homozygous Ptgs1 mutant mice survive well, have no gastric pathology, and show less indomethacin-induced gastric ulceration than wild-type mice, even though their gastric prostaglandin E2 levels are about 1% of wild type. The homozygous mutant mice have reduced platelet aggregation and a decreased inflammatory response to arachidonic acid, but not to tetradecanoyl phorbol acetate. Ptgs1 homozygous mutant females mated to homozygous mutant males produce few live offspring. COX-1-deficient mice provide a useful model to distinguish the physiological roles of COX-1 and COX-2.

Genetic control of blood pressure and the angiotensinogen locus.

Variants of the human angiotensinogen gene have been linked in some studies to increased circulating angiotensinogen levels and essential hypertension. To test for direct causality between genotypes at the angiotensinogen locus and blood pressures, we have studied mice carrying zero, one, two, three, or four functional copies of the murine wild-type angiotensinogen gene (Agt) at its normal chromosomal location. Plasma angiotensinogen levels increase progressively, although not linearly, from zero in the zero-copy animals to 145% of normal in the four-copy animals. Mice of all genotypes are normal at birth, but most zero-copy animals die before weaning. The kidneys of the zero-copy animals show pathological changes as adults, but the kidneys are normal in the other genotypes. One adult zero-copy male tested was fertile. The blood pressures of the one-copy through four-copy animals show significant and almost linear increases of approximately 8 mmHg per gene copy despite their normal compensatory mechanisms being intact. These results establish a direct causal relationship between Agt genotypes and blood pressures.

Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia.

Studies by various investigators have indicated that elevated levels of plasma homocyst(e)ine are strongly associated with the occurrence of occlusive vascular diseases. With the eventual aim of determining whether or not elevated plasma homocyst(e)ine concentrations are directly causative of cardiovascular diseases, we have generated mice that are moderately and severely homocyst(e)inemic. Homologous recombination in mouse embryonic stem cells was used to inactivate the cystathionine beta-synthase [L-serine hydrolyase (adding homocysteine), EC 4.2.1.22] gene. Homozygous mutants completely lacking cystathionine beta-synthase were born at the expected frequency from matings of heterozygotes, but they suffered from severe growth retardation and a majority of them died within 5 weeks after birth. Histological examination showed that the hepatocytes of homozygotes were enlarged, multinucleated, and filled with microvesicular lipid droplets. Plasma homocyst(e)ine levels of the homozygotes were approximately 40 times normal. These mice, therefore, represent a model for severe homocyst(e)inemia resulting from the complete lack of cystathionine beta-synthase. Heterozygous mutants have approximately 50% reduction in cystathionine beta-synthase mRNA and enzyme activity in the liver and have twice normal plasma homocyst(e)ine levels. Thus, the heterozygous mutants are promising for studying the in vivo role of elevated levels of homocyst(e)ine in the etiology of cardiovascular diseases.