Four-kilobase sequence of the mouse CNP gene directs spatial and temporal expression of lacZ in transgenic mice

The gene encoding 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) is one of the earliest myelin genes to be expressed in the brain. It is expressed at basal levels in some non-neural tissues but at much higher levels in the nervous system, and its relevance and mechanism are unknown. Using transgenic mice, we examined the expression pattern conferred by a 4-kilobase (-kb) 5'-flanking sequence of the mouse CNP gene coupled to the bacterial lacZ reporter gene. Here we report that this 4-kb fragment contains sufficient information to direct expression of the transgene to the tissue and/or cell type, in which CNP is normally expressed. In the central nervous system (CNS), CNP-lacZ expression was regulated in a temporal manner, consistent with endogenous CNP expression. Transgene expression was detected in embryonic brain and spinal cord in immature oligodendrocytes, and it significantly increased with age. In adult mice, beta-galactosidase activity (which appeared to be oligodendrocyte specific) was found essentially in white matter areas of the CNS. Moreover, the transgene was expressed in peripheral nervous system, testis, and thymus-tissues that normally express CNP. Taken together, our results provide strong evidence that cis-acting regulatory elements, necessary to direct spatial and temporal expression of the transgene in oligodendrocytes, are located within the 4-kb 5'-flanking sequence of the mouse CNP gene. This promoter could be a valuable tool to target specific expression of other transgenes to oligodendrocytes, and may provide important new insights into myelination or dysmyelination.     

A 6.1 kb 5' upstream region of the mouse tryptophan hydroxylase gene directs expression of E. coli lacZ to major serotonergic brain regions and pineal gland in transgenic mice

Tryptophan hydroxylase (TPH) catalyzes the first step of serotonin biosynthesis in serotonergic neurons and neuroendocrine cells. Serotonin influences diverse vital physiological functions and is thought to play an important role in several human psychiatric disorders. To localize DNA element(s) important for serotonergic tissue-specific expression of TPH, 6.1 kb of the 5' flanking region of the mouse TPH gene was fused to the coding region of the E. coli lacZ gene, and expression of the resulting fusion gene was analyzed in transgenic mice. The 6.1 kb of 5' flanking sequence was able to direct the expression of a lacZ reporter gene to serotonergic tissues in six lines of transgenic mice. A high level of lacZ expression in transgenic mice carrying the fusion gene was detected in the pineal gland as well as a moderate level of lacZ expression in serotonergic brain regions such as the median and dorsal raphe nuclei, the nuclei raphe magnus and raphe pallidus. In contrast, a smaller 5' flanking sequence of 1.1 kb directed no detectable serotonergic tissue-specific lacZ expression in five lines of transgenic mice. These results presented in this paper suggest first that DNA elements critical to serotonergic tissue-specific expression reside between -6.1 kb and -1.1 kb of 5' flanking region of the mouse TPH gene, but second that this region confers a restricted tissue-specific expression.     

The immediate 5'-flanking region of the rat phenylalanine hydroxylase-encoding gene

We have characterized the immediate (465 bp) 5'-flanking region of the rat phenylalanine hydroxylase (PAH)-encoding gene. This sequence shows considerable similarity to the 5'-flanking region of the human PAH gene [Konecki et al., Biochemistry 31 (1992) 8363-8368]. Both sequences lack obvious TATA elements; however, putative regulatory sites, including a potential cyclic AMP-response element and glucocorticoid response elements, are present.     

A developmental study of novH gene expression in human central nervous system

The expression pattern of the human nephroblastoma overexpressed (novH) gene in the fetal human central nervous system was examined by in situ hybridization using digoxigenin-labeled novH-specific riboprobes. In the spinal cord, the nov-expressing neurons were first detected both in the ventral region at 16 weeks of gestation (G16W) and in the dorsal region at G38W. In the medulla, nov-expressing neurons were detected in the principal nucleus of the inferior olive, the hypoglossal nucleus and the dorsal motor nucleus of vagus at G16W. Nov-positive neurons were detected at G28W in the nucleus of the spinal tract of the trigeminal and cuneate nucleus, and at G38W in the abducens nucleus of pons, the red nucleus and the substantia nigra of the midbrain, the ventral posterolateral and the mediodorsal thalamic nucleus. A strong labeling was also detected in the striatum of the cerebrum and the cerebral cortex of the parietal lobe. These data established that novH is mainly expressed in somato-motor neurons in the lower central nervous system at early developmental stages and in the higher central nervous system at later stages, suggesting that nov may play an important role in neuronal differentiation.     

5 alpha-reductase isozymes in the central nervous system

The enzyme 5 alpha-reductase (5 alpha-R) activates several delta 4-3keto steroids to more potent derivatives which may also acquire new biological actions. Testosterone gives rise to the most potent natural androgen dihydrotestosterone (DHT), and progesterone to dihydroprogesterone (DHP), a precursor of the endogenous anxiolytic/anesthetic steroid tetrahydroprogesterone (THP). Two isoforms of 5 alpha-R, with a limited degree of homology, different biochemical properties and distinct tissue distribution have been cloned: 5 alpha-R type 1 and type 2. In androgen-dependent structures DHT is almost exclusively formed by 5 alpha-R type 2; 5 alpha-R type 1 is widely distributed in the body, with the highest levels in the liver, and may be involved in steroid catabolism. In the brain, the roles of the two isozymes are still largely unknown. This brief review will summarize recent experimental data from our laboratory which try to assign possible functional roles to the process of 5 alpha-reduction, and to the two 5 alpha-R isoforms in the CNS.     

Regulation of neuronal plasticity in the central nervous system by phosphorylation and dephosphorylation

Neuronal plasticity can be defined as adaptive changes in structure and function of the nervous system, an obvious example of which is the capacity to remember and learn. Long-term potentiation and long-term depression are the experimental models of memory in the central nervous system (CNS), and have been frequently utilized for the analysis of the molecular mechanisms of memory formation. Extensive studies have demonstrated that various kinases and phosphatases regulate neuronal plasticity by phosphorylating and dephosphorylating proteins essential to the basic processes of adaptive changes in the CNS. These proteins include receptors, ion channels, synaptic vesicle proteins, and nuclear proteins. Multifunctional kinases (cAMP-dependent protein kinase, Ca2+/phospholipid-dependent protein kinase, and Ca2+/calmodulin-dependent protein kinases) and phosphatases (calcineurin, protein phosphatases 1, and 2A) that specifically modulate the phosphorylation status of neuronal-signaling proteins have been shown to be required for neuronal plasticity. In general, kinases are involved in upregulation of the activity of target substrates, and phosphatases downregulate them. Although this rule is applicable in most of the cases studied, there are also a number of exceptions. A variety of regulation mechanisms via phosphorylation and dephosphorylation mediated by multiple kinases and phosphatases are discussed.     

The 5alpha-reductase in the central nervous system: expression and modes of control

The present paper will summarize two important aspects of the interactions between steroids and the brain, which have recently been studied in the authors' laboratory. In particular the paper will consider data on: (1) the significance of the two isoforms of the 5alpha-R during brain ontogenesis and development, and (2) the cross-talk between glial and neuronal elements, particularly in relation to the metabolism of sex hormones. (1) The data obtained have shown that the 5alpha-R type 1 enzyme is constitutively expressed in the rat CNS at all stages of brain development. Moreover, the expression of the 5alpha-R type 1 is similar in males and in females, and does not appear to be controlled by androgens. The gene expression of the 5alpha-R type 2 is totally different. This isoform appears to be expressed in the rat brain almost exclusively in the late fetal/early post-natal life and is controlled by testosterone. (2) The present data show that two cell lines derived respectively from a rat glioma (C6 cell line) and from a human astrocytoma (1321N1 cell line) are able to convert testosterone and progesterone into their corresponding 5alpha-reduced metabolites dihydrotestosterone and dihydroprogesterone. The possibility that secretory products of normal and tumoral brain cells might be able to influence steroid metabolism occurring in the two glial cell lines previously mentioned has been considered.     

Replacement by homologous recombination of the minK gene with lacZ reveals restriction of minK expression to the mouse cardiac conduction system

Surgical approaches to the inner ear with hearing preservation have valuable implications for neurotologic surgery. In a previous study in guinea pigs, we demonstrated that click-evoked auditory brain stem responses (ABRs) were preserved after transection and plugging of 1 or more semicircular canals but were lost after entering the vestibule (Smouha EE, et al. Otolaryngol Head Neck Surg 1996; 114:777-784). A limitation of that study was that click-evoked ABR might not represent the function of the entire cochlea. In this study we used tone-burst ABR to determine thresholds across a broad range of frequencies (2 to 24 kHz) before and after surgical entry into the labyrinth at the lateral semicircular canal, ampulla, and vestibule. Serial measurements were made, and a sham surgery group was used as a control. The results obtained with tone-burst ABR generally agreed with those previously obtained with click stimuli. Toneburst ABR thresholds were similar across most frequencies tested. Transection of the lateral semicircular canal resulted in preservation of ABR thresholds. Ampullectomy had a variable effect on ABR thresholds. Vestibulotomy usually resulted in substantial hearing loss. We conclude that hearing can be preserved across a range of frequencies after selective surgery of the inner ear. Surgical entry into the membranous labyrinth near the vestibule is the critical factor contributing to hearing loss after partial labyrinthectomy.     

Cloning and characterization of the 5'-flanking region of the human cardiotrophin-1 gene

To enable the analysis of the regulation of the human cardiotrophin-1 gene expression, the 5'-flanking region of the human cardiotrophin-1 gene was cloned and sequenced. Data bank search revealed several cis- active DNA elements (SP1, CREB, C/EBP, AP1 and AP-2 like and GATA) in the proximal 1.1 kb region. Six nested 5-'terminal deletion mutants from -1091/+39 to -218/+39 were fused to a luciferase reportergene and proved to be functionally active after transfection into COS-7 cells.     

Regulation of human erythropoietin gene induction by upstream flanking sequences in transgenic mice

Human erythropoietin (Epo) gene expression is inducible by hypoxia or anaemia in the kidney and liver. Previous transgenic mouse experiments have demonstrated that sequences required for Epo gene induction in the kidney reside in a 7 8 kb Barn HI fragment located 6 kb upstream of the gene. To sublocalize these sequences, we performed Desoxyribonuclease I (DNAse I) mapping studies using transgenic mice which carried this DNA fragment. These studies revealed a DNAse I hypersensitive site (DNAse I HS) located 4 6 kb from the upstream end of the 7.8 kb fragment in anaemic kidney and liver samples. Sequence analysis of the region encompassing the DNAse I HS revealed an element with remarkable homology to the 3' Epo gene hypoxia-inducible enhancer. This suggested the presence of an additional regulatory element that contributes to the control of hypoxia-inducible Epo gene expression in kidney and liver. We constructed transgenic mice containing the human Epo gene linked to either the 5 kb upstream or 2.5 kb downstream portion of the 7.8kb fragment. Inducible expression was limited to the liver. Thus, neither fragment was alone sufficient to confer kidney inducible expression. These findings indicate that sequences more than 8.5 kb upstream of the Epo gene are required for kidney-specific induction. They suggest that either those sequences reside in an 0.3 kb Hind III fragment located between the 5 kb and the 2.5 kb fragments or that sequences in the 5 kb or 0.3 kb fragments must interact with sequences in the 2.5 kb fragment to allow Epo gene induction in the kidney.     

Functional expression of the human angiotensinogen gene in transgenic mice

The renin-angiotensin system is a major determinant of arterial pressure and volume homeostasis in mammals through the actions of angiotensin II, the proteolytic digestion product of angiotensinogen. Molecular genetic studies in several human populations have revealed genetic linkage between the angiotensinogen gene and both hypertension and increased plasma angiotensinogen. Transgenic mice were generated with a human angiotensinogen genomic clone to develop an animal model to examine tissue- and cell-specific expression of the gene and to determine if overexpression of angiotensinogen results in hypertension. Human angiotensinogen mRNA was expressed in transgenic mouse liver, kidney, heart, adrenal gland, ovary, brain, and white and brown adipose tissue and, in kidney, was exclusively localized to epithelial cells of the proximal convoluted tubules. Plasma levels of human angiotensinogen were approximately 150-fold higher in transgenic mice than that found normally in human plasma. The blood pressure of mice bearing the human angiotensinogen gene was normal but infusion of a single bolus dose of purified human renin resulted in a transient increase in blood pressure of approximately 30 mm Hg within 2 min. These results suggest that abnormalities in the angiotensinogen gene resulting in increased circulating levels of angiotensinogen could potentially contribute in part to the pathogenesis of essential hypertension.