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.

Prefrontal D1 dopamine signaling is necessary for temporal expectation during reaction time performance.

Responses during a simple reaction time task are influenced by temporal expectation, or the ability to anticipate when a stimulus occurs in time. Here, we test the hypothesis that prefrontal D1 dopamine signaling is necessary for temporal expectation during simple reaction time task performance. We depleted dopamine projections to the medial prefrontal circuits by infusing 6-hydroxidopamine, a selective neurotoxin, into the ventral tegmental area (VTA) of rats, and studied their performance on a simple reaction time task with two delays. VTA dopamine depletion did not change movements or learning of the reaction time task. However, VTA dopamine-depleted animals did not develop delay-dependent speeding of reaction times, suggesting that mesocortical dopamine signaling is required for temporal expectation. Next, we manipulated dopamine signaling within the medial prefrontal cortex using local pharmacology. We found that SCH23390, a D1-type dopamine receptor antagonist, specifically attenuated delay-dependent speeding, while sulpiride, a D2-type receptor antagonist, did not. These data suggest that prefrontal D1 dopamine signaling is necessary for temporal expectation during performance of a simple reaction time task. Our findings provide insight into temporal processing of the prefrontal cortex, and how dopamine signaling influences prefrontal circuits that guide goal-directed behavior.

Long-term safety of recombinant human growth hormone in children.

BACKGROUND: Between 1985 and 2006, the National Cooperative Growth Study (NCGS) monitored the safety and efficacy of recombinant human growth hormone (rhGH) in 54,996 children. METHODS: Enrolled patients were followed until rhGH discontinuation. Investigators submitted adverse event reports for targeted events or those potentially rhGH-related. RESULTS: Early concerns about de novo leukemia in patients without risk factors have not been substantiated--three observed vs. 5.6 expected in age-matched general population based on years at risk [standard incidence ratio (SIR), 0.54; 95% confidence interval (CI), 0.11-1.58]. De novo malignancies (intracranial and extracranial) were not significantly increased in patients without risk factors (29 confirmed vs. 26 expected; SIR, 1.12; 95% CI, 0.75-1.61). Second neoplasms occurred in 49 patients, of whom 37 had irradiation for their initial tumors (including five of 16 retinoblastoma patients, three of whom had bilateral retinoblastoma) consistent with an increased risk with rhGH. Thirty-three patients developed type 1 diabetes mellitus (DM) (37 expected; SIR, 0.90; 95% CI, 0.62-1.26). Type 2 DM and nonspecified DM were reported in 20 and eight patients, respectively. Two deaths were reported in patients with Prader-Willi syndrome and five deaths from aortic dissection in patients with Turner syndrome. In patients with organic GH deficiency and idiopathic panhypopituitarism, 11 events of acute adrenal insufficiency occurred, including four deaths, consistent with a reported increased risk for adrenal insufficiency in hypopituitary patients with or without rhGH treatment. CONCLUSION: After more than 20 yr, leukemia, a major safety issue initially believed associated with GH, has not been confirmed, but other signals, including risk of second malignancies in patients previously treated with irradiation, have been detected or confirmed through the NCGS. These data further clarify the events associated with rhGH and, although confirming a favorable overall safety profile, they also highlight specific populations at potential risk.

Combined haploinsufficiency of SF-1 and GATA4 does not reveal a genetic interaction in mouse gonadal development.

The nuclear receptor steroidogenic factor 1 (SF-1 or NR5A1) and the zinc finger protein GATA4 mediate key events in the early steps of gonadal development and sex differentiation, presumably by activating the expression of essential target genes. An important SF-1 target in male sex differentiation is the gene encoding the anti-Müllerian hormone (AMH), which induces regression of the Müllerian ducts in the developing male embryo. In cell transfection studies, there is apparent cooperation between GATA4 and SF-1 in the regulation of both human and mouse Amh promoters. We hypothesized that compound haploinsufficiency of both SF-1 and GATA4, by reducing their synergism, might cause a more severe phenotype than that seen in mice that were heterozygous for either SF-1 or Gata4 alone. Surprisingly, in adult and embryonic mice, compound haploinsufficiency of SF-1 and GATA4 caused no gonadal or reproductive abnormalities beyond those seen in SF-1(+/-) mice. Thus, although cooperation between SF-1 and GATA4 very likely is important for regulation of their target genes, such synergy was not revealed in our in vivo studies of gonadal development and function.

Pituitary-specific knockout of steroidogenic factor 1.

Knockout mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1) revealed its essential roles at multiple levels of endocrine development and function. These SF-1 knockout mice lacked adrenal glands and gonads, thereby manifesting adrenal insufficiency and sex reversal of their internal and external genitalia. Their pituitary gonadotropes failed to express several markers of normal differentiated function, and they lacked a specific hypothalamic nucleus, the ventromedial hypothalamic nucleus (VMH). Using the Cre-loxP system, we generated mice whose gene encoding SF-1 was inactivated specifically in the anterior pituitary. These pituitary-specific SF-1 knockout mice were sterile and never matured sexually. Their gonads weighed only approximately 5% of the weight of wild-type gonads. SF-1 immunoreactivity was absent in the anterior pituitary but was unaffected in the adrenal cortex, validating the selectivity of the gene targeting strategy. Consistent with an important role of SF-1 in gonadotropes, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were markedly decreased in the pituitary-specific SF-1 knockout mice. The pituitary-specific SF-1 knockout mice are a novel genetic model of hypogonadotropic hypogonadism and establish essential roles of SF-1 in gonadotropin expression.

Hypomorphic phenotype in mice with pituitary-specific knockout of steroidogenic factor 1.

The bacteriophage Cre recombinase provides a powerful approach for tissue-specific gene inactivation. Using a Cre transgene driven by the common alpha subunit of glycoprotein hormones (alphaGSU-Cre), we have previously inactivated steroidogenic factor 1 (SF-1) in the anterior pituitary, causing hypogonadotropic hypogonadism with sexual infantilism, sterility, and severe gonadal hypoplasia. We now explore the molecular mechanisms underlying a hypomorphic gonadal phenotype in mice carrying two floxed SF-1 alleles (F/F) relative to mice carrying one recombined and one floxed allele (F/R). Because their Cre-mediated disruption of the locus encoding SF-1 was less efficient, alphaGSU-Cre, F/F mice retained some gonadotropin-expressing cells in the anterior pituitary, thereby stimulating some gonadal function. This novel in vivo model for exploring the effects of differing levels of gonadotropins on gonadal development highlights the need for careful genotype-phenotype comparisons in studies using Cre recombinase to produce tissue-specific knockouts.

Approaches to define the role of SF-1 at different levels of the hypothalamic-pituitary-steroidogenic organ axis.

Targeted gene disruption has produced knockout mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 knockout mice lacked adrenal glands and gonads, resulting in adrenocortical insufficiency and sex reversal of their internal and external genitalia. They also had impaired expression of pituitary gonadotropins and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at multiple levels of the hypothalamic-pituitary-steroidogenic tissue axis. Using the Cre-loxP system, we now have generated mice in which SF-1 is inactivated selectively in the anterior pituitary. These pituitary-specific SF-1 knockout mice were sterile and failed to exhibit sexual maturation. Histologically, their gonads were markedly hypoplastic, weighing only approximately 5% of the gonads of wild-type mice. Consistent with an important role of SF-1 in gonadotropes, there were no cells in the pituitary gland that expressed either follicle-stimulating hormone (FSH) or luteinizing hormone (LH). These pituitary-specific SF-1 knockout mice are a novel genetic model of hypogonadotropic hypogonadism and establish essential roles of SF-1 in gonadotropin expression.

Genes essential for early events in gonadal development.

The acquisition of a sexually dimorphic phenotype is a critical event in mammalian development. The basic underlying principle of sexual development is that genetic sex--determined at fertilization by the presence or absence of the Y chromosome--directs the embryonic gonads to differentiate into either testes or ovaries. Thereafter, hormones produced by the testes direct the developmental program that leads to male sexual differentiation. In the absence of testicular hormones, the female pathway of sexual differentiation occurs. Recent studies have defined key roles in gonadal development for two transcription factors: Wilms' tumor suppressor 1 (WT1) and steroidogenic factor 1 (SF-1). After presenting a brief overview of gonadal development and sexual differentiation, this chapter reviews the studies that led to the isolation and characterization of WT1 and SF-1, and then discusses how interactions between these two genes may mediate their key roles in a common developmental pathway.

TFIIAalpha/beta-like factor is encoded by a germ cell-specific gene whose expression is up-regulated with other general transcription factors during spermatogenesis in the mouse.

TFIIAalpha/beta-like factor (ALF) is a testis-specific counterpart of the large subunit of human general transcription factor TFIIA. Northern analysis shows that ALF mRNA first appears in mouse testis at Postnatal Day 14. Similarly, expression of the general transcription factors TBP, TRF2, TFIIAalpha/beta, TFIIAgamma, and TFIIIB(90) is also increased beginning at Postnatal Day 14, suggesting that there is a coordinated induction of many general transcription factors during male germ cell differentiation. Analysis of male germ cells separated by Staput sedimentation shows that ALF is present in pachytene spermatocytes and haploid spermatids. In addition, in situ hybridization experiments with adult mouse testis shows that ALF is present in haploid spermatids. Searches of the human genome sequence database using the basic local alignment search tool reveal that the ALF and TFIIAalpha/beta(GTF2A1) genes are both composed of nine exons, whereas the TFIIAgamma (GTF2A2) gene is composed of five exons. Furthermore, nucleotide and amino acid comparisons among human and mouse ALF, TFIIAalpha/beta, and TFIIAgamma cDNA sequences show that ALF has diverged more rapidly than either TFIIAalpha/beta or TFIIAgamma. Finally, the ALF and SBLF (Stoned B-Like Factor) sequences present in the chimeric SALF cDNA are both present on human chromosome 2, and an analysis of the corresponding genes suggests a model for the formation of SALF.

SF-1: a critical mediator of steroidogenesis.

Studies in knockout mice have established that the orphan nuclear receptor steroidogenic factor 1 (SF-1) plays essential roles in the development and function of the primary steroidogenic organs. These SF-1 knockout mice lacked adrenal glands and gonads, causing adrenocortical insufficiency and sex reversal of their internal and external genitalia. They also had impaired expression of pituitary gonadotropins and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at all three levels of the hypothalamic-pituitary-steroidogenic organ axis. Ongoing experiments are directed at developing methods to inactivate SF-1 in a tissue-specific manner.

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.

Steroidogenic factor 1 (SF1) is essential for pituitary gonadotrope function.

Knockout mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF1) exhibit a complex endocrine phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and absence of the ventromedial hypothalamic nucleus (VMH). These multiple defects complicate efforts to delineate primary versus secondary effects of SF1 deficiency in different tissues, such that its direct role in gonadotropes remains uncertain. To define this role, we have expressed Cre recombinase driven by the promoter region of the common alpha subunit of glycoprotein hormones (alpha GSU), thereby inactivating a loxP-modified SF1 locus in the anterior pituitary gland. Although pituitary-specific SF1 knockout mice were fully viable, they were sterile and failed to develop normal secondary sexual characteristics. Their adrenal glands and VMH appeared normal histologically, but their testes and ovaries were severely hypoplastic. alpha GSU-Cre, loxP mice had normal levels of most pituitary hormones, but had markedly decreased expression of LH and FSH. Treatment with exogenous gonadotropins stimulated gonadal steroidogenesis, inducing germ cell maturation in males and follicular and uterine maturation in females--establishing that the gonads can respond to gonadotropins. The pituitary-specific SF1 knockout mice are a novel genetic model of hypogonadotropic hypogonadism that establishes essential role(s) of SF1 in pituitary gonadotropes.

Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice.

Steroidogenic acute regulatory protein (StAR) is essential for adrenal and gonadal steroidogenesis, stimulating the translocation of cholesterol to the inner mitochondrial membrane where steroidogenesis commences. StAR mutations in humans cause congenital lipoid adrenal hyperplasia (lipoid CAH), an autosomal recessive condition with severe deficiencies of all classes of steroid hormones. We previously described StAR knockout mice that mimic many features of lipoid CAH patients. By keeping StAR knockout mice alive with corticosteroid replacement, we now examine the temporal effects of StAR deficiency on the structure and function of steroidogenic tissues. The adrenal glands, affected most severely at birth, exhibited progressive increases in lipid deposits with aging. The testes of newborn StAR knockout mice contained scattered lipid deposits in the interstitial region, presumably in remnants of fetal Leydig cells. By 8 weeks of age, the interstitial lipid deposits worsened considerably and were associated with Leydig cell hyperplasia. Despite these changes, germ cells in the seminiferous tubules appeared intact histologically, suggesting that the StAR knockout mice retained some capacity for androgen biosynthesis. Sperm maturation was delayed, and the germ cells exhibited histological features of apoptosis, consistent with suboptimal androgen production. Immediately after birth, the ovaries of StAR knockout mice appeared normal. After the time of normal puberty, however, prominent lipid deposits accumulated in the interstitial region, accompanied by marked luteinization of stromal cells and incomplete follicular maturation that ultimately culminated in premature ovarian failure. These studies provide the first systematic evaluation of the developmental consequences of StAR deficiency in the various steroidogenic organs.

Steroidogenic factor 1 (SF-1) is essential for ovarian development and function.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) was identified originally as a key regulator of the tissue-specific expression of the cytochrome P450 steroid hydroxylases. Hints at considerably broader roles for SF-1 came from analyses of its expression pattern in mouse embryos. As anticipated, SF-1 was expressed in the adrenal glands and gonads from their early stages of development. Surprisingly, SF-1 also was expressed outside of the primary steroidogenic tissues in the anterior pituitary and hypothalamus. SF-1 knockout mice dramatically confirmed its multiple essential roles in vivo. These mice lacked adrenal glands and gonads, leading to adrenocortical insufficiency and male-to-female sex reversal of their internal and external genitalia. SF-1 knockout mice also had impaired pituitary expression of gonadotropins and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at all three levels of the hypothalamic-pituitary-gonadal axis. With some focus on the ovary, this review summarizes experiments that have defined essential roles of SF-1 in endocrine development, and highlights important areas for future studies.

The LIM homeobox gene Lhx9 is essential for mouse gonad formation.

During mammalian embryonic development, the ovaries and testes develop from somatic cells of the urogenital ridges as indifferent gonads, harbouring primordial germ cells that have migrated there. After sex determination of the gonads, the testes produce testosterone and anti-Mullerian hormone which mediate male sexual differentiation, and the female developmental pathway ensues in their absence. Here we show that transcripts of the LIM homeobox gene Lhx9 are present in urogenital ridges of mice at embryonic day 9.5; later they localize to the interstitial region as morphological differentiation occurs. In mice lacking Lhx9 function, germ cells migrate normally, but somatic cells of the genital ridge fail to proliferate and a discrete gonad fails to form. In the absence of testosterone and anti-Mullerian hormone, genetically male mice are phenotypically female. The expression of steroidogenic factor 1 (Sf1), a nuclear receptor essential for gonadogenesis, is reduced to minimal levels in the Lhx9-deficient genital ridge, indicating that Lhx9 may lie upstream of Sf1 in a developmental cascade. Unlike mice lacking other genes that mediate early stages of gonadogenesis, Lhx9 mutants do not exhibit additional major developmental defects. Thus, LHX9 mutations may underlie certain forms of isolated gonadal agenesis in humans.

Approaches to define the role of SF-1 at different levels of the hypothalamic-pituitary-steroidogenic organ axis.

Targeted gene disruption has produced knockout mice globally deficient in the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 knockout mice lacked adrenal glands and gonads and consequently exhibited adrenocortical insufficiency and sex reversal of their internal and external genitalia. They also had impaired expression of gonadtropins in the anterior pituitary gonadotropes and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at all three levels of the hypothalamic-pituitary-steroidogenic organ axis. Ongoing experiments are directed at using evolving techniques for tissue-specific gene inactivation to define the roles of SF-1 within discrete sites of the hypthalamic-pituitary-steroidogenic organ axis.

Developmental and hormonal regulation of murine scavenger receptor, class B, type 1.

The scavenger receptor, class B, type I (SR-BI), is the predominant receptor that supplies plasma cholesterol to steroidogenic tissues in rodents. We showed previously that steroidogenic factor-1 (SF-1) binds a sequence in the human SR-BI promoter whose integrity is required for high-level SR-BI expression in cultured adrenocortical tumor cells. We now provide in vivo evidence that SF-1 regulates SR-BI. During mouse embryogenesis, SR-BI mRNA was initially expressed in the genital ridge of both sexes and persisted in the developing testes but not ovary. This sexually dimorphic expression profile of SR-BI expression in the gonads mirrors that of SF-1. No SR-BI mRNA was detected in the gonadal ridge of day 11.5 SF-1 knockout embryos. Both SR-BI and SF-1 mRNA were expressed in the cortical cells of the nascent adrenal glands. These studies directly support SF-1 participating in the regulation of SR-BI in vivo. We examined the effect of cAMP on SR-BI mRNA and protein in mouse adrenocortical (Y1-BS1) and testicular carcinoma Leydig (MA-10) cells. The time courses of induction were strikingly similar to those described for other cAMP- and SF-1-regulated genes. Addition of lipoproteins reduced SR-BI expression in Y1-BS1 cells, an effect that was reversed by administration of cAMP analogs. SR-BI mRNA and protein were expressed at high levels in the adrenal glands of knockout mice lacking the steroidogenic acute regulatory protein; these mice have extensive lipid deposits in the adrenocortical cells and high circulating levels of ACTH. Taken together, these studies suggest that trophic hormones can override the suppressive effect of cholesterol on SR-BI expression, thus ensuring that steroidogenesis is maintained during stress.

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.

Genes essential for early events in gonadal development.

The acquisition of a sexually dimorphic phenotype is a critical event in mammalian development. The basic underlying principle of sexual development is that genetic sex-determined at fertilization by the presence or absence of the Y chromosome--directs the embryonic gonads to differentiate into either testes or ovaries. Thereafter, hormones produced by the testes direct the developmental program that leads to male sexual differentiation. In the absence of testicular hormones, the female pathway of sexual differentiation occurs. Recent studies have defined key roles in gonadal development for two transcription factors: Wilms' tumor suppressor 1 (WT1) and steroidogenic factor 1 (SF-1). After presenting a brief overview of gonadal development and sexual differentiation, this paper reviews the studies that led to the isolation and characterization of WT1 and SF-1, and then discusses how interactions between these two genes may mediate their key roles in a common developmental pathway.