Delta-frequency stimulation of cerebellar projections can compensate for schizophrenia-related medial frontal dysfunction.

Schizophrenia involves abnormalities in the medial frontal cortex that lead to cognitive deficits. Here we investigate a novel strategy to normalize medial frontal brain activity by stimulating cerebellar projections. We used an interval timing task to study elementary cognitive processing that requires both frontal and cerebellar networks that are disrupted in patients with schizophrenia. We report three novel findings. First, patients with schizophrenia had dysfunctional delta rhythms between 1-4 Hz in the medial frontal cortex. We explored cerebellar-frontal interactions in animal models and found that both frontal and cerebellar neurons were modulated during interval timing and had delta-frequency interactions. Finally, delta-frequency optogenetic stimulation of thalamic synaptic terminals of lateral cerebellar projection neurons rescued timing performance as well as medial frontal activity in a rodent model of schizophrenia-related frontal dysfunction. These data provide insight into how the cerebellum influences medial frontal networks and the role of the cerebellum in cognitive processing.

Non-Mendelian inheritance of mosquito susceptibility to infection with Brugia malayi and Brugia pahangi.

The mode of inheritance of susceptibility or refractoriness of insect vectors to medically important pathogens such as those causing malaria or filariasis is usually believed to follow normal Mendelian laws and to involve a single pair of alleles. In this report, experiments are described that demonstrate another mode of inheritance of mosquito susceptibility to filarial parasites. Crosses were made between susceptibile and refractory species of the Aedes Scutellaris complex, and the hybrid and backcross progeny were tested for susceptibility to infection by Brugia malayi and Brugia pahangi. The data indicate that inheritance follows a non-Mendelian pattern indicative of extrachromosomal factors inherited through the maternal parent.

Nonsense mutation causing steroid 21-hydroxylase deficiency.

We determined the sequence of a mutant CYP21B gene isolated from a patient with the severe, "salt-wasting" form of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. Codon 318 in this gene is changed from CAG, encoding glutamine, to TAG, a nonsense codon. This is predicted to result in a completely nonfunctional enzyme due to premature termination of translation. In addition, when the cloned mutant gene was transfected into mouse Y1 adrenal cells, the resulting mRNA levels were decreased compared with transfected normal CYP21B genes. This mutation was carried by 3 of 20 unrelated patients with 21-hydroxylase deficiency alleles as determined by hybridization with a specific oligonucleotide probe. This mutation is also seen in the normal CYP21A pseudogene, so that its presence in the abnormal CYP21B gene may be the result of a gene conversion event.

The orphan nuclear receptor NGFI-B regulates expression of the gene encoding steroid 21-hydroxylase.

As part of its trophic action to maintain the steroidogenic capacity of adrenocortical cells, corticotropin (ACTH) increases the transcription of the cytochrome P-450 steroid hydroxylase genes, including the gene encoding steroid 21-hydroxylase (21-OHase). We previously identified several promoter elements that regulate 21-OHase gene expression in mouse Y1 adrenocortical tumor cells. One of these elements, located at nucleotide -65, closely resembles the recognition sequence of the orphan nuclear receptor NGFI-B, suggesting that NGFI-B regulates this essential steroidogenic enzyme. To explore this possibility, we first used in situ hybridization to demonstrate high levels of NGFI-B transcripts in the adrenal cortex of the adult rat. In cultured mouse Y1 adrenocortical cells, treatment with ACTH, the major regulator of 21-OHase transcription, rapidly increased NGFI-B expression. Gel mobility shift and DNase I footprinting experiments showed that recombinantly expressed NGFI-B interacts specifically with the 21-OHase -65 element and identified one complex formed by Y1 extracts and the 21-OHase -65 element that contains NGFI-B. Expression of NGFI-B significantly augmented the activity of the intact 21-OHase promoter, while mutations of the -65 element that abolish NGFI-B binding markedly diminished NGFI-B-mediated transcriptional activation. Specific mutations of NGFI-B shown previously to impair either DNA binding or transcriptional activation diminished the effect of NGFI-B coexpression on 21-OHase expression. Finally, an oligonucleotide containing the NGFI-B response element conferred ACTH response to a core promoter from the prolactin gene, showing that this element is sufficient for ACTH induction. Collectively, these results identify a cellular promoter element that is regulated by NGFI-B and implicate NGFI-B in the transcriptional induction of 21-OHase by ACTH.

Expression of steroidogenic factor 1 and Wilms' tumour 1 during early human gonadal development and sex determination.

The transcription factors SF-1 and WT1 play pivotal roles in mammalian gonadal development and sexual differentiation. In human embryos, both SF-1 and WT1 are expressed when the indifferent gonadal ridge first forms at 32 days post-ovulation. As the sex cords develop - providing morphological evidence of testis differentiation - SF-1 localises predominantly to developing Sertoli cells in the sex cords, whereas WT1 retains a broader pattern of expression. Later, SF-1 localises predominantly to steroidogenic Leydig cells, and WT1 localises to the sex cords. In the ovary, SF-1 and WT1 transcripts persist in the gonadal ridge from the earliest developmental stages throughout the critical period of sex determination. These studies, which delineate for the first time the sequential expression profiles of SF-1 and WT1 during human gonadal development, provide a framework for understanding human sex reversal phenotypes associated with their mutations.

The roles of the nuclear receptor steroidogenic factor 1 in endocrine differentiation and development.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) has emerged as a critical determinant of adrenal and gonadal differentiation, development, and function. SF-1 was initially isolated as a positive regulator of the cytochrome P450 steroid hydroxylases in the adrenal glands and gonads; developmental analyses subsequently showed that SF-1 was also expressed in the diencephalon and anterior pituitary, suggesting additional roles in endocrine function. Analyses of knockout mice deficient in SF-1 revealed multiple abnormalities, including adrenal and gonadal agenesis, male to female sex reversal of the internal genitalia, impaired gonadotrope function, and absence of the ventromedial hypothalamic nucleus. Taken together, these results implicate SF-1 as a global regulator within the hypothalamic-pituitary-gonadal axis and the adrenal cortex.

Transcriptional regulation of the adrenal steroidogenic enzymes.

Corticosteroid biosynthesis requires the concerted action of a related group of cytochrome P450 steroid hydroxylases. The genes encoding these steroid hydroxylases exhibit two distinct levels of transcriptional regulation: selective expression in steroidogenic cells and induction in response to trophic hormones. With respect to cell-selective expression, recent studies have identified a nuclear receptor protein expressed only in steroidogenic cells that is postulated to regulate the expression of all cytochrome P450 steroid hydroxylases through common promoter elements. In contrast, the coordinate responses of these genes to trophic hormones are not readily explained by a unifying mechanism, and their hormone responsive expression probably involves multiple promoter elements.

Steroidogenic factor I, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor I.

We proposed that a cell-selective regulatory protein coordinately regulates the expression of three enzymes that are required for the biosynthesis of corticosteroids: cholesterol side chain cleavage enzyme, steroid 21-hydroxylase, and the aldosterone synthase isozyme of steroid 11 beta-hydroxylase. In this report, we identify a 53-kilodalton protein, termed steroidogenic factor 1 (SF-1), that interacts with the related promoter elements from these steroidogenic enzymes, and we isolate and characterize a cDNA that very likely encodes this protein. We first showed that nuclear extracts from bovine adrenal glands interact with the mouse steroidogenic regulatory elements, forming complexes indistinguishable from those produced by nuclear extracts from mouse Y1 adrenocortical cells. These bovine adrenal extracts were subjected to sequential ion exchange and affinity chromatography to yield a highly enriched preparation of SF-1. The predominant protein in the affinity-purified preparation comigrated with shift activity and had a mol wt of 53,000; UV cross-linking experiments demonstrated directly that this 53-kilodalton protein interacted with the steroidogenic regulatory element. Even with this marked enrichment, affinity-purified SF-1 bound six steroidogenic regulatory elements. These results support strongly the model that a steroidogenic cell-selective protein interacts with related promoter elements from three steroidogenic enzymes to regulate their coordinate expression. The recognition sequence of SF-1 closely resembles those of nuclear hormone receptor family members, suggesting that SF-1 may belong to this supergene family. By screening a Y1 cell cDNA library with the DNA-binding region of the H-2RIIBP nuclear hormone receptor cDNA, we isolated a cDNA that is selectively expressed in steroidogenic cells. When expressed as a glutathione S-transferase fusion protein in Escherichia. coli, the protein encoded by this cDNA interacts with all six related steroidogenic regulatory elements with a binding specificity indistinguishable from that of SF-1. Surprisingly, the sequence of the putative DNA-binding domain of this cDNA matches exactly the corresponding sequence of the mouse homolog of the Drosophila transcription factor fushi tarazu-factor I. The demonstration that a member of the nuclear hormone receptor family interacts with the steroidogenic regulatory elements provides intriguing insights into possible mechanisms by which these essential genes are regulated.

Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases.

As an initial step toward understanding its role in steroidogenesis, we studied the developmental profile of steroidogenic factor-1 (SF-1), a nuclear receptor that regulates the steroid hydroxylases. SF-1 transcripts first appear on embryonic day 9 (E9) in the urogenital ridge, the probable source of steroidogenic cells of both adrenals and gonads. By E11, after the adrenals and gonads are clearly separate, SF-1 transcripts are detected throughout the adrenal primordium. Thereafter, adrenal expression of SF-1 localizes to the cortex. Consistent with its proposed role in regulating cholesterol side-chain cleavage enzyme (SCC), SF-1 is expressed before SCC. During the sexually undifferentiated stage of gonadal development (E9-E12), all embryos express SF-1 in the genital ridge. As testicular cords form in males, SF-1 transcripts are diffusely expressed throughout the testis, whereas SCC mRNA is limited to the interstitium. These differences between SF-1 and SCC reflect SF-1 expression by Sertoli cells, as shown by Northern blotting and in situ hybridization. In contrast to its persistent expression in the embryonic testis, SF-1 transcripts disappear from the ovary between E13.5-E16.5, reappearing only during late gestation (E18.5). Thus, expression of SF-1 in the embryonic gonad is sexually dimorphic. Coupled with the demonstration of SF-1 mRNA in Sertoli cells, these data suggest that SF-1 plays a role in gonadal development distinct from regulating the steroidogenic enzymes. Additionally, SF-1 is expressed in the embryonic forebrain, implying a role in neural development.

Steroidogenic factor 1 is the essential transcript of the mouse Ftz-F1 gene.

Targeted disruption of the mouse Ftz-F1 gene, which encodes the orphan nuclear receptors steroidogenic factor 1 (SF-1) and embryonal long terminal repeat-binding protein (ELP), established that this gene is essential for development of the primary steroidogenic tissues and for male sexual differentiation. Associated with these dramatic developmental abnormalities, all Ftz-F1-disrupted mice died in the immediate postnatal period and had very low glucocorticoid levels. In this report, we show that treatment with corticosteroids markedly prolonged survival of the Ftz-F1-disrupted mice, proving that steroid hormone deficiency causes their death. We also generated SF-1-specific knockout mice with a targeting construct that specifically disrupted the SF-1 coding sequence without impairing the ELP protein. The phenotype of the SF-1-specific knockout mice was indistinguishable from that observed in Ftz-F1-disrupted mice that lack both SF-1 and ELP. Taken together, these results indicate that SF-1 is the Ftz-F1-encoded protein that is required for multiple aspects of endocrine development and for postnatal survival.

Cholesterol side-chain cleavage cytochrome P450 gene expression in the primitive gut of the mouse embryo does not require steroidogenic factor 1.

In situ hybridization studies reveal novel sites of expression of cholesterol side-chain cleavage cytochrome P450 (P450scc) during murine embryonic development. In addition to fetal adrenals and testes, P450scc transcripts localize in situ to the primitive gut and to a subset of unidentified cells in the dermal mesenchyme of embryonic skin. In the gut, transcripts are most abundant in luminal epithelia of the hindgut, which will form the colon. P450scc transcript abundance at these novel sites is a fraction of that in fetal adrenals or testes, suggesting a local rather than an endocrine function. Immunocytochemical analyses localize P450scc protein to the fetal hindgut, indicating that the transcripts are translated in vivo. RNA isolated from microdissected embryonic hindgut and skin was reverse transcribed and amplified by polymerase chain reaction. DNA sequence analyses of polymerase chain reaction products confirmed that specific hybridization in situ represents authentic P450scc gene (Cyp11A) transcripts and that 3 beta-hydroxysteroid dehydrogenase/delta 5-->delta 4-isomerase transcripts are also present, demonstrating the potential of these fetal tissues to produce pregnenolone and progesterone. P450scc transcripts are also detectable by in situ hybridization in primitive gut and skin of Fushi tarazu factor 1 null mice, which lack the nuclear receptor steroidogenic factor 1, proving that steroidogenic factor 1 is not required for steroid hydroxylase gene expression at these sites. The capacity for C21 steroid biosynthesis in primitive gut and skin during organogenesis raises the question whether local production of steroid hormones may be required for normal cellular growth and differentiation of these tissues during embryogenesis.

Identification of promoter elements in the mouse 21-hydroxylase (Cyp21) gene that require a functional cyclic adenosine 3',5'-monophosphate-dependent protein kinase.

The constitutive and cAMP-induced expression of the mouse steroid 21-hydroxylase gene (Cyp21) are impaired in adrenal cell mutants harboring mutations in cAMP-dependent protein kinase (cAMPdPK). These requirements for a functional cAMPdPK have been mapped to the proximal 330 basepairs of the Cyp21 promoter. This study attempts to identify specific promoter elements of Cyp21 that require cAMPdPK for constitutive activity by comparing their abilities to enhance the expression of a reporter gene in Y1 adrenocortical tumor cells and Y1 Kin mutants defective in cAMPdPK activity. As determined in transient transfection assays, Cyp21 promoter elements at -65, -140, -170, -210, and -280 each enhanced the expression of a human GH reporter gene in parent Y1 cells. The relative order of effectiveness of each of these elements was: -170 >> -280 > -140 > -65 > or = -210. The -170 element was 25-fold more effective in enhancing gene expression from the reporter construct in Y1 cells than in Kin mutant cells; the elements at -65, -140, and -210 were 3-fold more effective in Y1 cells than in Kin mutant cells; the -280 element was equally effective in the parent and Kin mutant clones. These studies suggest that the promoter elements at -170, -65, -140, and -210 mediate the requirement for a functional cAMPdPK in the expression of Cyp21. As determined by gel mobility shift assays with these elements, the dependence of the Cyp21 promoter elements on a functional cAMP-dependent protein kinase did not result from decreased expression or binding affinities of their respective DNA-binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

cis modification of the steroid 21-hydroxylase gene prevents its expression in the Y1 mouse adrenocortical tumor cell line.

The Y1 mouse adrenocortical tumor cell line retains the ability to synthesize and secrete steroids, but does not express steroid 21-hydroxylase (C21) and, therefore, does not produce 21-hydroxylated steroids. In this investigation the mechanisms underlying the loss of C21 activity in the Y1 cell line were explored. A 9-kilobase BglII fragment containing the C21 gene was cloned from the Y1 genome. This genomic clone directed the synthesis of C21 transcripts and 21-hydroxylated steroid products when transfected back into the Y1 cell line. As determined by restriction endonuclease digestions with MspI and HpaII, enzymes that distinguish between unmethylated and methylated CCGG sites, the endogenous C21 gene was extensively methylated in Y1 adrenal cells and in cells from other mouse tissues that do not normally express this gene. In contrast, the C21 gene was hypomethylated in primary cultures of mouse adrenal cells which normally synthesize large amounts of C21. The cloned C21 gene transfected into Y1 cells initially was unmethylated, but became extensively methylated with prolonged culture of the cells; prolonged culture of these transfectants also resulted in a loss of C21 expression. Loss of C21 expression in Y1 transfectants, however, temporally preceded the extensive methylation of the transfected C21 gene. Furthermore, treatment of Y1 cells with 5-azacytidine caused a demethylation of the endogenous C21 gene, but did not result in the recovery of C21 expression. These results indicate that Y1 cells contain a functional C21 gene that has been silenced by a reversible cis-modification event.(ABSTRACT TRUNCATED AT 250 WORDS)

A shared promoter element regulates the expression of three steroidogenic enzymes.

The adrenal cortex of the mouse coordinately expresses three cytochrome P450 enzymes that are required for the biosynthesis of corticosteroids: cholesterol side-chain cleavage enzyme (SCC), steroid 21-hydroxylase (21-OHase), and steroid 11 beta-hydroxylase (11 beta-OHase). Within their 5'-flanking regions, we previously identified six elements containing variations of an AGGTC motif that regulated expression in mouse Y1 adrenocortical cells: 21-OHase elements at -210, -140, and -65; SCC elements at -70 and -40; and an 11 beta-OHase element at -310. We demonstrate here that all six elements interact with the same, or closely related, DNA-binding protein(s). First, these elements all formed complexes of similar mobility in gel shift assays, suggesting that they interacted with protein(s) of similar size. Additional larger complexes were seen with those probes containing exact AGGTCA sequences. Second, competition experiments confirmed that the factor(s) interacting with different elements had closely related or identical recognition specificities. Finally, indistinguishable profiles of shift activities were seen upon fractionation of nuclear proteins over sequential chromatographic columns. Collectively, these results suggest that related elements interact with a shared protein to regulate three essential steroidogenic enzymes. An AGGTCA sequence motif comprises the response element for several members of the nuclear hormone receptor family. Oligonucleotide competitions and specific effects of antisera in gel shift assays implicated chicken ovalbumin upstream promoter-transcription factor in the formation of the larger complexes seen with the elements containing exact AGGTCA sequences. Therefore, this member of the nuclear hormone receptor family also may regulate the expression of the adrenal steroidogenic enzymes.

The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus.

The nuclear receptor steroidogenic factor 1 (SF-1) regulates the biosynthesis of the two essential mediators of male sexual differentiation, androgens and Müllerian-inhibiting substance, and is required for adrenal and gonadal development and gonadotropin expression. SF-1 is also expressed in the embryonic ventral diencephalon, subsequently localizing to the ventromedial hypothalamic nucleus, a region important for reproductive behavior. Mice lacking SF-1 secondary to targeted disruption of the Ftz-F1 gene had normal numbers and location of GnRH neurons but exhibited grossly impaired ventromedial hypothalamic nucleus structure. Despite their apparently normal GnRH neurons, treatment of Ftz-F1-disrupted mice with GnRH restored pituitary gonadotropin expression. These studies define SF-1's essential role within a discrete hypothalamic nucleus previously linked to reproduction.

Expression profiles of SF-1, DAX1, and CYP17 in the human fetal adrenal gland: potential interactions in gene regulation.

Cytochrome P450 17alpha-hydroxylase/17-20 lyase (P450(C17)) is a critical branchpoint enzyme for steroid hormone biosynthesis. During human gestation, P450(C17) is required for the production of dehydroepiandrostenedione sulfate by the fetal adrenal cortex and for testicular production of androgens that mediate male sexual differentiation. In this study, we investigate the regulation of the human CYP17 gene by two orphan nuclear receptors, steroidogenic factor 1 (SF-1) and DAX1. In human embryos, SF-1 and DAX1 are expressed throughout the developing adrenal cortex from its inception at 33 days post conception (dpc). In contrast, P450(C17) expression, which commences between 41 and 44 dpc, is limited to the fetal zone. The 5'-flanking region of the human CYP17 gene contains three functional SF-1 elements that collectively mediate a > or =25-fold induction of promoter activity by SF-1. In constructs containing all three functional SF-1 elements, DAX1 inhibited this activation by > or =55%. In the presence of only one or two SF-1 elements, DAX1 inhibition was lost even though SF-1 transactivation persisted. These data suggest that efficient repression of SF-1-mediated activation of the human CYP17 gene by DAX1 requires multiple SF-1 elements. Opposing effects of SF-1 and DAX1 may fine tune the differential responses of various SF-1 target genes in different endocrine tissues.

Identification and characterization of two upstream elements that regulate adrenocortical expression of steroid 11 beta-hydroxylase.

We have studied the mechanisms that regulate the expression of the mouse gene encoding steroid 11 beta-hydroxylase (11 beta-OHase), a steroidogenic cytochrome P450 enzyme that is expressed only in the adrenal cortex. DNase I footprinting and gel-mobility shift analyses revealed potential regulatory elements at -370 and -310 in the 11 beta-OHase promoter region. To determine the contributions of these elements to expression, we altered their sequences by site-selected mutagenesis and studied promoter activity after transfection into Y1 mouse adrenocortical tumor cells. Mutation of either element markedly decreased basal promoter activity but did not affect the response to treatment with 8-bromo cAMP. These experiments thus document the functional roles of these elements, within the context of the intact promoter, in constitutive expression of 11 beta-OHase. Moreover, addition of either of these elements to p-40GH, a 5'-deletion plasmid containing 11 beta-OHase sequences from -40 to +8 upstream of a growth hormone reporter gene, significantly increased promoter activity but did not confer cAMP responsiveness. Finally, increased expression was seen after transfection of Y1 derivatives deficient in cAMP-dependent protein kinase, indicating that neither element required cAMP-dependent protein kinase activity. These studies thus define two regulatory elements that play important roles in 11 beta-OHase expression.

Characterization of the mouse FTZ-F1 gene, which encodes a key regulator of steroid hydroxylase gene expression.

The cytochrome P450 steroid hydroxylases are coordinately regulated by steroidogenic factor 1 (SF-1), a protein expressed selectively in steroidogenic cells. Based on its expression in steroidogenic tissues and DNA-binding specificity, we isolated a putative SF-1 cDNA from an adrenocortical cDNA library. As evidence that this cDNA encodes SF-1, we now show that it is selectively expressed in steroidogenic cells, that an antiserum against its protein product specifically abolishes the SF-1-related gel-shift complex, and that its coexpression increases promoter activity of the 21-hydroxylase 5'-flanking region in transfection experiments. Sequence analyses of the SF-1 cDNA revealed that it is the mouse homolog of fushi tarazu factor I (FTZ-F1), a nuclear receptor that regulates the fushi tarazu homeobox gene in Drosophila. A second FTZ-F1 homolog, embryonal long terminal repeat-binding protein (ELP), was recently isolated from embryonal carcinoma cells. SF-1 and ELP cDNAs are virtually identical for 1017 base pairs, including putative DNA-binding domains, but diverge at their 5'- and 3'-ends. One genomic clone contained both SF-1- and ELP-specific sequences, confirming their origin from a single gene. Characterization of this gene defined shared exons encoding common regions and alternative promoters and 3'-exons leading to differences between the two FTZ-F1 transcripts. We used in situ hybridization with transcript-specific probes to study the ontogeny of SF-1 and ELP expression. ELP transcripts were not detected from embryonic day 8 to adult, consistent with its previous isolation from embryonal carcinoma cells and its postulated role in early embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)

Variegated expression of a mouse steroid 21-hydroxylase/beta- galactosidase transgene suggests centripetal migration of adrenocortical cells.

5'-Flanking sequences (6.4 kb) of the mouse steroid 21-hyrodxylase (21-OHase) A gene linked to a LacZ reporter gene directed appropriate cell-specific expression in cultured Y1 adrenocortical tumor cells and in the adrenal cortex of transgenic mice. The transgene expression initiated at the same stage of adrenal development as the endogenous 21-OHase gene (embryonic day 11.5). Although the endogenous 21-OHase gene is expressed throughout the adrenal cortex, the 21-OHase/beta-gal transgene showed a strikingly variegated pattern of adrenocortical expression in all 10 transgene-expressing mouse lines examined. This presents as radial stripes of beta-gal staining transcending the classical zonal structure of the adrenal cortex but paralleling the columnar arrangement of cells of the zona fasciculata on the centripetal organization of the adrenocortical blood supply. To the extent that the variegated pattern of 21-OHase/beta-gal transgene expression depicts adrenocortical cell lineage, these results suggest that all cells within an individual stripe have a common clonal origin; the radial pattern of clonally derived cells argues that cellular migration maintains the adult adrenocortical cell population. Adrenal glands of developing embryos also exhibited a variegated pattern of 21-OHase/beta-gal transgene expression. However, this presented as islands of beta-gal reporter staining within the developing gland, suggesting that the rapid embryonic adrenal growth phase, which precedes the establishment of the classic adrenocortical zonal structure, may be governed by cellular mechanisms distinct from those responsible for maintenance of the adult adrenocortical cell population.