Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene

The formation of estrogens from C19 steroids is catalyzed by aromatase cytochrome P450 (P450arom), the product of the cyp19 gene. The actions of estrogen include dimorphic anatomical, functional, and behavioral effects on the development of both males and females, considerations that prompted us to examine the consequences of deficiency of aromatase activity in mice. Mice lacking a functional aromatase enzyme (ArKO) were generated by targeted disruption of the cyp19 gene. Male and female ArKO mice were born with the expected Mendelian frequency from F1 parents and grew to adulthood. Female ArKO mice at 9 weeks of age displayed underdeveloped external genitalia and uteri. Ovaries contained numerous follicles with abundant granulosa cells and evidence of antrum formation that appeared arrested before ovulation. No corpora lutea were present. Additionally the stroma were hyperplastic with structures that appeared to be atretic follicles. Development of the mammary glands approximated that of a prepubertal female. Examination of male ArKO mice of the same age revealed essentially normal internal anatomy but with enlargement of the male accessory sex glands because of increased content of secreted material. The testes appeared normal. Male ArKO mice are capable of breeding and produce litters of approximately average size. Whereas serum estradiol levels were at the limit of detection, testosterone levels were elevated, as were the levels of follicle-stimulating hormone and luteinizing hormone. The phenotype of these animals differs markedly from that of the previously reported ERKO mice, in which the estrogen receptor α is deleted by targeted disruption.

Tertiary hypothyroidism and hyperglycemia in mice with targeted disruption of the thyrotropin-releasing hormone gene

Thyrotropin-releasing hormone (TRH) is a brain hypothalamic hormone that regulates thyrotropin (TSH) secretion from the anterior pituitary and is ubiquitously distributed throughout the brain and other tissues including pancreas. To facilitate studies into the role of endogenous TRH, we have used homologous recombination to generate mice that lack TRH. These TRH−/− mice are viable, fertile, and exhibit normal development. However, they showed obvious hypothyroidism with characteristic elevation of serum TSH level and diminished TSH biological activity. Their anterior pituitaries exhibited an apparent decrease in TSH immunopositive cells that was not due to hypothyroidism. Furthermore, this decrease could be reversed by TRH, but not thyroid hormone replacement, suggesting a direct involvement of TRH in the regulation of thyrotrophs. The TRH−/− mice also exhibited hyperglycemia, which was accompanied by impaired insulin secretion in response to glucose. These findings indicate that TRH−/− mice provide a model of exploiting tertiary hypothyroidism, and that TRH gene abnormalities cause disturbance of insulin secretion resulting in marked hyperglycemia.

Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein provides insights into congenital lipoid adrenal hyperplasia

An essential component of regulated steroidogenesis is the translocation of cholesterol from the cytoplasm to the inner mitochondrial membrane where the cholesterol side-chain cleavage enzyme carries out the first committed step in steroidogenesis. Recent studies showed that a 30-kDa mitochondrial phosphoprotein, designated steroidogenic acute regulatory protein (StAR), is essential for this translocation. To allow us to explore the roles of StAR in a system amenable to experimental manipulation and to develop an animal model for the human disorder lipoid congenital adrenal hyperplasia (lipoid CAH), we used targeted gene disruption to produce StAR knockout mice. These StAR knockout mice were indistinguishable initially from wild-type littermates, except that males and females had female external genitalia. After birth, they failed to grow normally and died from adrenocortical insufficiency. Hormone assays confirmed severe defects in adrenal steroids—with loss of negative feedback regulation at hypothalamic–pituitary levels—whereas hormones constituting the gonadal axis did not differ significantly from levels in wild-type littermates. Histologically, the adrenal cortex of StAR knockout mice contained florid lipid deposits, with lesser deposits in the steroidogenic compartment of the testis and none in the ovary. The sex-specific differences in gonadal involvement support a two-stage model of the pathogenesis of StAR deficiency, with trophic hormone stimulation inducing progressive accumulation of lipids within the steroidogenic cells and ultimately causing their death. These StAR knockout mice provide a useful model system in which to determine the mechanisms of StAR’s essential roles in adrenocortical and gonadal steroidogenesis.

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse)

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

Mouse model of Sanfilippo syndrome type B produced by targeted disruption of the gene encoding α-N-acetylglucosaminidase

The Sanfilippo syndrome type B is an autosomal recessive disorder caused by mutation in the gene (NAGLU) encoding α-N-acetylglucosaminidase, a lysosomal enzyme required for the stepwise degradation of heparan sulfate. The most serious manifestations are profound mental retardation, intractable behavior problems, and death in the second decade. To generate a model for studies of pathophysiology and of potential therapy, we disrupted exon 6 of Naglu, the homologous mouse gene. Naglu−/− mice were healthy and fertile while young and could survive for 8–12 mo. They were totally deficient in α-N-acetylglucosaminidase and had massive accumulation of heparan sulfate in liver and kidney as well as secondary changes in activity of several other lysosomal enzymes in liver and brain and elevation of gangliosides GM2 and GM3 in brain. Vacuolation was seen in many cells, including macrophages, epithelial cells, and neurons, and became more prominent with age. Although most vacuoles contained finely granular material characteristic of glycosaminoglycan accumulation, large pleiomorphic inclusions were seen in some neurons and pericytes in the brain. Abnormal hypoactive behavior was manifested by 4.5-mo-old Naglu−/− mice in an open field test; the hyperactivity that is characteristic of affected children was not observed even in younger mice. In a Pavlovian fear conditioning test, the 4.5-mo-old mutant mice showed normal response to context, indicating intact hippocampal-dependent learning, but reduced response to a conditioning tone, perhaps attributable to hearing impairment. The phenotype of the α-N-acetylglucosaminidase-deficient mice is sufficiently similar to that of patients with the Sanfilippo syndrome type B to make these mice a good model for study of pathophysiology and for development of therapy.

Impairing follicle-stimulating hormone (FSH) signaling in vivo: Targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance

Pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone stimulate the gonads by regulating germ cell proliferation and differentiation. FSH receptors (FSH-Rs) are localized to testicular Sertoli cells and ovarian granulosa cells and are coupled to activation of the adenylyl cyclase and other signaling pathways. Activation of FSH-Rs is considered essential for folliculogenesis in the female and spermatogenesis in the male. We have generated mice lacking FSH-R by homologous recombination. FSH-R-deficient males are fertile but display small testes and partial spermatogenic failure. Thus, although FSH signaling is not essential for initiating spermatogenesis, it appears to be required for adequate viability and motility of the sperms. FSH-R-deficient females display thin uteri and small ovaries and are sterile because of a block in folliculogenesis before antral follicle formation. Although the expression of marker genes is only moderately altered in FSH-R −/− mice, drastic sex-specific changes are observed in the levels of various hormones. The anterior lobe of the pituitary gland in females is enlarged and reveals a larger number of FSH- and thyroid-stimulating hormone (TSH)-positive cells. The phenotype of FSH-R −/− mice is reminiscent of human hypergonadotropic ovarian dysgenesis and infertility.

Targeted disruption of the murine dihydrolipoamide dehydrogenase gene (Dld) results in perigastrulation lethality

The Dld gene product, known as dihydrolipoamide dehydrogenase or the E3 component, catalyzes the oxidation of dihydrolipoyl moieties of four mitochondrial multienzyme complexes: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain α-ketoacid dehydrogenase, and the glycine cleavage system. Deficiency of E3 activity in humans results in various degrees of neurological dysfunction and organic acidosis caused by accumulation of branched-chain amino acids and lactic acid. In this study, we have introduced a null mutation into the murine Dld gene (Dldtm1mjp). The heterozygous animals are shown to have approximately half of wild-type activity levels for E3 and all affected multienzyme complexes but are phenotypically normal. In contrast, the Dld−/− class dies prenatally with apparent developmental delay at 7.5 days postcoitum followed by resorption by 9.5 days postcoitum. The Dld−/− embryos cease to develop at a time shortly after implantation into the uterine wall when most of the embryos have begun to gastrulate. This null phenotype provides in vivo evidence for the requirement of a mitochondrial oxidative pathway during the perigastrulation period. Furthermore, the early prenatal lethal condition of the complete deficiency state may explain the low incidence of detectable cases of E3 deficiency in humans.

Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (ICa-L) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on ICa-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOSnull mice, with targeted disruption of the eNOS gene. Although both eNOSnull and wild-type (WT) ventricular myocytes exhibited similar increases in ICa-L in response to ISO, there was no measurable suppression of ICa-L by CCh in cells from eNOSnull mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOSnull myocytes. Also, unlike myocytes from WT animals, eNOSnull myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented ICa-L in eNOSnull cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K+ channel by CCh was unaffected in atrial and ventricular eNOSnull myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of ICa-L in cardiac myocytes.

Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD +/− mice yielded an abnormally low number of LCAD +/− and −/− offspring, indicating frequent gestational loss. LCAD −/− mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD −/− males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD −/− mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD in vivo.

Targeted disruption of the Kcnq1 gene produces a mouse model of Jervell and Lange– Nielsen Syndrome

KCNQ1 encodes KCNQ1, which belongs to a family of voltage-dependent K+ ion channel proteins. KCNQ1 associates with a regulatory subunit, KCNE1, to produce the cardiac repolarizing current, IKs. Loss-of-function mutations in the human KCNQ1 gene have been linked to Jervell and Lange–Nielsen Syndrome (JLNS), a disorder characterized by profound bilateral deafness and a cardiac phenotype. To generate a mouse model for JLNS, we created a line of transgenic mice that have a targeted disruption in the Kcnq1 gene. Behavioral analysis revealed that the Kcnq1−/− mice are deaf and exhibit a shaker/waltzer phenotype. Histological analysis of the inner ear structures of Kcnq1−/− mice revealed gross morphological anomalies because of the drastic reduction in the volume of endolymph. ECGs recorded from Kcnq1−/− mice demonstrated abnormal T- and P-wave morphologies and prolongation of the QT and JT intervals when measured in vivo, but not in isolated hearts. These changes are indicative of cardiac repolarization defects that appear to be induced by extracardiac signals. Together, these data suggest that Kcnq1−/− mice are a potentially valuable animal model of JLNS.

Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1

DAX-1 [dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1] is an orphan nuclear receptor that represses transcription by steroidogenic factor-1 (SF-1), a factor that regulates expression of multiple steroidogenic enzymes and other genes involved in reproduction. Mutations in the human DAX1 gene (also known as AHC) cause the X-linked syndrome AHC, a disorder that is associated with hypogonadotropic hypogonadism also. Characterization of Dax1-deficient male mice revealed primary testicular defects that included Leydig cell hyperplasia (LCH) and progressive degeneration of the germinal epithelium, leading to infertility. In this study, we investigated the effect of Dax1 disruption on the expression profile of various steroidogenic enzyme genes in Leydig cells isolated from Dax1-deficient male mice. Expression of the aromatase (Cyp19) gene, which encodes the enzyme that converts testosterone to estradiol, was increased significantly in the Leydig cells isolated from mutant mice, whereas the expression of other proteins (e.g., StAR and Cyp11a) was not altered. In in vitro transfection studies, DAX-1 repressed the SF-1-mediated transactivation of the Cyp19 promoter but did not inhibit the StAR or Cyp11a promoters. Elevated Cyp19 expression was accompanied by increased intratesticular levels of estradiol. Administration of tamoxifen, a selective estrogen-receptor modulator, restored fertility to the Dax1-deficient male mice and partially corrected LCH, suggesting that estrogen excess contributes to LCH and infertility. Based on these in vivo and in vitro analyses, aromatase seems to be a physiologic target of Dax-1 in Leydig cells, and increased Cyp19 expression may account, in part, for the infertility and LCH in Dax1-deficient mice.

Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.

Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in the postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in developing brain. Cdk5 purified from nervous tissue phosphorylates neuronal cytoskeletal proteins including neurofilament proteins and microtubule-associated protein tau in vitro. These findings indicate that Cdk5 may have unique functions in neuronal cells, especially in the regulation of phosphorylation of cytoskeletal molecules. We report here generation of Cdk5(-/-) mice through gene targeting and their phenotypic analysis. Cdk5(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of Cdk5(-/-) mice lack cortical laminar structure and cerebellar foliation. In addition, the large neurons in the brain stem and in the spinal cord show chromatolytic changes with accumulation of neurofilament immunoreactivity. These findings indicate that Cdk5 is an important molecule for brain development and neuronal differentiation and also suggest that Cdk5 may play critical roles in neuronal cytoskeleton structure and organization.

Targeted disruption of the arylsulfatase B gene results in mice resembling the phenotype of mucopolysaccharidosis VI.

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase B (ASB), which is involved in degradation of dermatan sulfate and chondroitin 4-sulfate. A MPS VI mouse model was generated by targeted disruption of the ASB gene. Homozygous mutant animals exhibit ASB enzyme deficiency and elevated urinary secretion of dermatan sulfate. They develop progressive symptoms resembling those of MPS VI in humans. Around 4 weeks of age facial dysmorphia becomes overt, long bones are shortened, and pelvic and costal abnormalities are observed. Major alterations in bone formation with perturbed cartilaginous tissues in newborns and widened, perturbed, and persisting growth plates in adult animals are seen. All major parenchymal organs show storage of glycosaminoglycans preferentially in interstitial cells and macrophages. Affected mice are fertile and mortality is not elevated up to 15 months of age. This mouse model will be a valuable tool for studying pathogenesis of MPS VI and may help to evaluate therapeutical approaches for lysosomal storage diseases.

Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects.

At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.