Impaired generation of mature neurons due to extended expression of Tlx by repressing Sox2 transcriptional activity.

As a master regulator of the dynamic process of adult neurogenesis, timely expression and regulation of the orphan nuclear receptor Tailless (Tlx) is essential. However, there is no study yet to directly investigate the essential role of precise spatiotemporal expressed Tlx. Here, we generated a conditional gain of Tlx expression transgenic mouse model, which allowed the extended Tlx expression in neural stem cells (NSCs) and their progeny by mating with a TlxCreERT2 mouse line. We demonstrate that extended expression of Tlx induced the impaired generation of mature neurons in adult subventricular zone and subgranular zone. Furthermore, we elucidated for the first time that this mutation decreased the endogenous expression of Sox2 by directly binding to its promoter. Restoration experiments further confirmed that Sox2 partially rescued these neuron maturation defects. Together, these findings not only highlight the importance of shutting-off Tlx on time in controlling NSC behavior, but also provide insights for further understanding adult neurogenesis and developing treatment strategies for neurological disorders.

The nuclear receptor tailless induces long-term neural stem cell expansion and brain tumor initiation.

Malignant gliomas are the most common primary brain tumors, and are associated with frequent resistance to therapy as well as poor prognosis. Here we demonstrate that the nuclear receptor tailless (Tlx), which in the adult is expressed exclusively in astrocyte-like B cells of the subventricular zone, acts as a key regulator of neural stem cell (NSC) expansion and brain tumor initiation from NSCs. Overexpression of Tlx antagonizes age-dependent exhaustion of NSCs in mice and leads to migration of stem/progenitor cells from their natural niche. The increase of NSCs persists with age, and leads to efficient production of newborn neurons in aged brain tissues. These cells initiate the development of glioma-like lesions and gliomas. Glioma development is accelerated upon loss of the tumor suppressor p53. Tlx-induced NSC expansion and gliomagenesis are associated with increased angiogenesis, which allows for the migration and maintenance of brain tumor stem cells in the perivascular niche. We also demonstrate that Tlx transcripts are overexpressed in human primary glioblastomas in which Tlx expression is restricted to a subpopulation of nestin-positive perivascular tumor cells. Our study clearly demonstrates how NSCs contribute to brain tumorgenesis driven by a stem cell-specific transcription factor, thus providing novel insights into the histogenesis and molecular pathogenesis of primary brain tumors.

The nuclear receptor tailless is required for neurogenesis in the adult subventricular zone.

The tailless (Tlx) gene encodes an orphan nuclear receptor that is expressed by neural stem/progenitor cells in the adult brain of the subventricular zone (SVZ) and the dentate gyrus (DG). The function of Tlx in neural stem cells of the adult SVZ remains largely unknown. We show here that in the SVZ of the adult brain Tlx is exclusively expressed in astrocyte-like B cells. An inducible mutation of the Tlx gene in the adult brain leads to complete loss of SVZ neurogenesis. Furthermore, analysis indicates that Tlx is required for the transition from radial glial cells to astrocyte-like neural stem cells. These findings demonstrate the crucial role of Tlx in the generation and maintenance of NSCs in the adult SVZ in vivo.

Inactivation of the gene for the nuclear receptor tailless in the brain preserving its function in the eye.

During embryogenesis, tailless, an orphan member of the nuclear receptor family, is expressed in the germinal zones of the brain and the developing retina, and is involved in regulating the cell cycle of progenitor cells. Consequently, a deletion of the tailless gene leads to decreased cell number with associated anatomical defects in the limbic system, the cortex and the eye. These structural abnormalities are associated with blindness, increased aggressiveness, poor performance in learning paradigms and reduced anxiousness. In order to assess the contribution of blindness to the behavioural changes, we established tailless mutant mice with intact visual abilities. We generated a mouse line in which the second exon of the tailless gene is flanked by loxP sites and crossed these animals with a transgenic line expressing the Cre recombinase in the neurogenic area of the developing brain, but not in the eye. The resulting animals have anatomically indistinguishable brains compared with tailless germline mutants, but are not blind. They are less anxious and much more aggressive than controls, like tailless germline mutants. In contrast to germline mutants, the conditional mutants are not impaired in fear conditioning. Furthermore, they show good performance in the Morris water-maze despite severely reduced hippocampal structures. Thus, the pathological aggressiveness and reduced anxiety found in tailless germline mutants are due to malformations caused by inactivation of the tailless gene in the brain, but the poor performance of tailless null mice in learning and memory paradigms is dependent on the associated blindness.

Definition of estrogen receptor pathway critical for estrogen positive feedback to gonadotropin-releasing hormone neurons and fertility.

The mechanisms through which estrogen regulates gonadotropin-releasing hormone (GnRH) neurons to control mammalian ovulation are unknown. We found that estrogen positive feedback to generate the preovulatory gonadotropin surge was normal in estrogen receptor beta knockout (ERbeta) mutant mice, but absent in ERalpha mutant mice. An ERalpha-selective compound was sufficient to generate positive feedback in wild-type mice. As GnRH neurons do not express ERalpha, estrogen positive feedback upon GnRH neurons must be indirect in nature. To establish the cell type responsible, we generated a neuron-specific ERalpha mutant mouse line. These mice failed to exhibit estrogen positive feedback, demonstrating that neurons expressing ERalpha are critical. We then used a GnRH neuron-specific Pseudorabies virus (PRV) tracing approach to show that the ERalpha-expressing neurons innervating GnRH neurons are located within rostral periventricular regions of the hypothalamus. These studies demonstrate that ovulation is driven by estrogen actions upon ERalpha-expressing neuronal afferents to GnRH neurons.

Hypothalamic 3',5'-cyclic adenosine monophosphate response element-binding protein loss causes anterior pituitary hypoplasia and dwarfism in mice.

The principal regulation of body growth is via a cascade of hormone signals emanating from the hypothalamus, by release of GHRH, which then directs the somatotroph cells of the pituitary to release GH into the blood stream. This in turn leads to activation of signal transducer and activator of transcription 5-dependent expression of genes such as IGF-I in hepatocytes, acid labile substance, and serine protease inhibitor 2.1, resulting in body growth. Here, using conditional cAMP response element binding protein (CREB) mutant mice, we show that loss of the CREB transcription factor in the brain, but not the pituitary, results in reduced postnatal growth consistent with dwarfism caused by GH deficiency. We demonstrate that although there appears to be no significant impact upon the expression of GHRH mRNA in CREB mutant mice, the amount of GHRH peptide is reduced. These findings show that CREB is required for the efficient production of GHRH in hypothalamus, in addition to its previously reported role in pituitary GH production and somatotroph expansion.

The epithelial glucocorticoid receptor is required for the normal timing of cell proliferation during mammary lobuloalveolar development but is dispensable for milk production.

Glucocorticoids have been shown to influence mammary gland function in vivo and to stimulate milk protein gene expression in vitro. Here, we describe the generation and analysis of a mouse model to study glucocorticoid receptor (GR, NR3C1) function in mammary epithelial cells. Using the Cre-loxP system, mutant mice were obtained in which the GR gene is specifically deleted in epithelial cells during lobuloalveolar development, leading to a complete loss of epithelial GR at the onset of lactation. Mice harboring the mammary-epithelial-specific GR mutation are able to nurse their litters until weaning. During pregnancy, however, GR deficiency delays lobuloalveolar development, leading to an incomplete epithelial penetration of the mammary fat pad that persists throughout lactation. We identified a reduced cell proliferation during lobuloalveolar development as reason for this delay. This reduction is compensated for by increased epithelial proliferation after parturition in the mutant glands. During lactation, GR-deficient mammary epithelium is capable of milk production and secretion. The expression of two milk proteins, namely whey acidic protein and beta-casein, during lactation was not critically affected in the absence of GR. We conclude that GR function is not essential for alveolar differentiation and milk production, but influences cell proliferation during lobuloalveolar development.

The Tlx gene regulates the timing of neurogenesis in the cortex.

The tailless (tlx) gene is a forebrain-restricted transcription factor. Tlx mutant animals exhibit a reduction in the size of the cerebral hemispheres and associated structures (Monaghan et al., 1997). Superficial cortical layers are specifically reduced, whereas deep layers are relatively unaltered (Land and Monaghan, 2003). To determine whether the adult laminar phenotype has a developmental etiology and whether it is associated with a change in proliferation/differentiation decisions, we examined the cell cycle and neurogenesis in the embryonic cortex. We found that there is a temporal and regional requirement for the Tlx protein in progenitor cells (PCs). Neurons prematurely differentiate at all rostrocaudal levels up to mid-neurogenesis in mutant animals. Heterozygote animals have an intermediate phenotype indicating there is a threshold requirement for Tlx in early cortical neurogenesis. Our studies indicate that PCs in the ventricular zone are sensitive to loss of Tlx in caudal regions only; however, PCs in the subventricular zone are altered at all rostrocaudal levels in tlx-deficient animals. Furthermore, we found that the cell cycle is shorter from embryonic day 9.5 in tlx-/- embryos. At mid-neurogenesis, the PC population becomes depleted, and late PCs have a longer cell cycle in tlx-deficient animals. Consequently, later generated structures, such as upper cortical layers, the dentate gyrus, and the olfactory bulbs, are severely reduced. These studies indicate that tlx is an essential intrinsic regulator in the decision to proliferate or differentiate in the developing forebrain.