A small population of hypothalamic neurons govern fertility: the critical role of VAX1 in GnRH neuron development and fertility maintenance.

Fertility depends on the correct maturation and function of approximately 800 gonadotropin-releasing hormone (GnRH) neurons in the brain. GnRH neurons are at the apex of the hypothalamic-pituitary-gonadal axis that regulates fertility. In adulthood, GnRH neurons are scattered throughout the anterior hypothalamic area and project to the median eminence, where GnRH is released into the portal vasculature to stimulate release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. LH and FSH then regulate gonadal steroidogenesis and gametogenesis. Absence of GnRH neurons or inappropriate GnRH release leads to infertility. Despite the critical role of GnRH neurons in fertility, we still have a limited understanding of the genes responsible for proper GnRH neuron development and function in adulthood. GnRH neurons originate in the olfactory placode then migrate into the brain. Homeodomain transcription factors expressed within GnRH neurons or along their migratory path are candidate genes for inherited infertility. Using a combined in vitro and in vivo approach, we have identified Ventral Anterior Homeobox 1 (Vax1) as a novel homeodomain transcription factor responsible for GnRH neuron maturation and fertility. GnRH neuron counts in Vax1 knock-out embryos revealed Vax1 to be required for the presence of GnRH-expressing cells at embryonic day 17.5 (E17.5), but not at E13.5. To localize the effects of Vax1 on fertility, we generated Vax1flox mice and crossed them with Gnrhcre mice to specifically delete Vax1 within GnRH neurons. GnRH staining in Vax1flox/flox:GnRHcre mice show a total absence of GnRH expression in the adult. We performed lineage tracing in Vax1flox/flox:GnRHcre:RosaLacZ mice which proved GnRH neurons to be alive, but incapable of expressing GnRH. The absence of GnRH leads to delayed puberty, hypogonadism and complete infertility in both sexes. Finally, using the immortalized model GnRH neuron cell lines, GN11 and GT1-7, we show that VAX1 is a direct regulator of Gnrh1 transcription by binding key ATTA sites within the Gnrh1 promoter. This study identifies VAX1 as a key transcription factor regulating GnRH expression and establishes VAX1 as a novel candidate gene implicated in heritable infertility

EFFECTS OF BANKING STRUCTURE ON THE ALLOCATION OF CREDIT TO NONMETROPOLITAN COMMUNITIES

Recent and proposed legislative changes encourage increases in multioffice banking activity. In this manuscript, the allocation of credit to nonmetropolitan communities in a branch banking state (Arizona) is compared to that in a unit banking-holding company state (Colorado). Rapidly growing nonmetropolitan areas have experienced increased lending activity under statewide branching relative to unit banking. Rural communities, which experienced slow or negative growth, had lower loan-to-deposit rations under branch banking than might have existed under unit banking. Therefore, conversion to branch banking may result in a reallocation of loanable funds within nonmetropolitan areas.Financial Economics,

A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models.

Gonadotropin-releasing hormone (GnRH), a neuropeptide released from a small population of neurons in the hypothalamus, is the central mediator of the hypothalamic-pituitary-gonadal axis, and is required for normal reproductive development and function. Evolutionarily conserved regulatory elements in the mouse, rat, and human Gnrh1 gene include three enhancers and the proximal promoter, which confer Gnrh1 gene expression specifically in GnRH neurons. In immortalized mouse hypothalamic GnRH (GT1-7) neurons, which show pulsatile GnRH release in culture, RNA sequencing and RT-qPCR revealed that expression of a novel long noncoding RNA at Gnrh1 enhancer 1 correlates with high levels of GnRH mRNA expression. In GT1-7 neurons, which contain a transgene carrying 3 kb of the rat Gnrh1 regulatory region, both the mouse and rat Gnrh1 enhancer-derived noncoding RNAs (GnRH-E1 RNAs) are expressed. We investigated the characteristics and function of the endogenous mouse GnRH-E1 RNA. Strand-specific RT-PCR analysis of GnRH-E1 RNA in GT1-7 cells revealed GnRH-E1 RNAs that are transcribed in the sense and antisense directions from distinct 5' start sites, are 3' polyadenylated, and are over 2 kb in length. These RNAs are localized in the nucleus and have a half-life of over 8 hours. In GT1-7 neurons, siRNA knockdown of mouse GnRH-E1 RNA resulted in a significant decrease in the expression of the Gnrh1 primary transcript and Gnrh1 mRNA. Over-expression of either the sense or antisense mouse GnRH-E1 RNA in immature, migratory GnRH (GN11) neurons, which do not express either GnRH-E1 RNA or GnRH mRNA, induced the transcriptional activity of co-transfected rat Gnrh1 gene regulatory elements, where the induction requires the presence of the rat Gnrh1 promoter. Together, these data indicate that GnRH-E1 RNA is an inducer of Gnrh1 gene expression. GnRH-E1 RNA may play an important role in the development and maturation of GnRH neurons

ESIM_DSN Web-Enabled Distributed Simulation Network

In this paper, the eSim(sup DSN) approach to achieve distributed simulation capability using the Internet is presented. With this approach a complete simulation can be assembled from component subsystems that run on different computers. The subsystems interact with each other via the Internet The distributed simulation uses a hub-and-spoke type network topology. It provides the ability to dynamically link simulation subsystem models to different computers as well as the ability to assign a particular model to each computer. A proof-of-concept demonstrator is also presented. The eSim(sup DSN) demonstrator can be accessed at http://www.jsc.draper.com/esim which hosts various examples of Web enabled simulations

Roles of binding elements, FOXL2 domains, and interactions with cJUN and SMADs in regulation of FSHβ.

We previously identified FOXL2 as a critical component in FSHβ gene transcription. Here, we show that mice deficient in FOXL2 have lower levels of gonadotropin gene expression and fewer LH- and FSH-containing cells, but the same level of other pituitary hormones compared to wild-type littermates, highlighting a role of FOXL2 in the pituitary gonadotrope. Further, we investigate the function of FOXL2 in the gonadotrope cell and determine which domains of the FOXL2 protein are necessary for induction of FSHβ transcription. There is a stronger induction of FSHβ reporter transcription by truncated FOXL2 proteins, but no induction with the mutant lacking the forkhead domain. Specifically, FOXL2 plays a role in activin induction of FSHβ, functioning in concert with activin-induced SMAD proteins. Activin acts through multiple promoter elements to induce FSHβ expression, some of which bind FOXL2. Each of these FOXL2-binding sites is either juxtaposed or overlapping with a SMAD-binding element. We determined that FOXL2 and SMAD4 proteins form a higher order complex on the most proximal FOXL2 site. Surprisingly, two other sites important for activin induction bind neither SMADs nor FOXL2, suggesting additional factors at work. Furthermore, we show that FOXL2 plays a role in synergistic induction of FSHβ by GnRH and activin through interactions with the cJUN component of the AP1 complex that is necessary for GnRH responsiveness. Collectively, our results demonstrate the necessity of FOXL2 for proper FSH production in mice and implicate FOXL2 in integration of transcription factors at the level of the FSHβ promoter

Chromatin status and transcription factor binding to gonadotropin promoters in gonadotrope cell lines.

BackgroundProper expression of key reproductive hormones from gonadotrope cells of the pituitary is required for pubertal onset and reproduction. To further our understanding of the molecular events taking place during embryonic development, leading to expression of the glycoproteins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), we characterized chromatin structure changes, imparted mainly by histone modifications, in model gonadotrope cell lines.MethodsWe evaluated chromatin status and gene expression profiles by chromatin immunoprecipitation assays, DNase sensitivity assay, and RNA sequencing in three developmentally staged gonadotrope cell lines, αT1-1 (progenitor, expressing Cga), αT3-1 (immature, expressing Cga and Gnrhr), and LβT2 (mature, expressing Cga, Gnrhr, Lhb, and Fshb), to assess changes in chromatin status and transcription factor access of gonadotrope-specific genes.ResultsWe found the common mRNA α-subunit of LH and FSH, called Cga, to have an open chromatin conformation in all three cell lines. In contrast, chromatin status of Gnrhr is open only in αT3-1 and LβT2 cells. Lhb begins to open in LβT2 cells and was further opened by activin treatment. Histone H3 modifications associated with active chromatin were high on Gnrhr in αT3-1 and LβT2, and Lhb in LβT2 cells, while H3 modifications associated with repressed chromatin were low on Gnrhr, Lhb, and Fshb in LβT2 cells. Finally, chromatin status correlates with the progressive access of LHX3 to Cga and Gnrhr, followed by PITX1 binding to the Lhb promoter.ConclusionOur data show the gonadotrope-specific genes Cga, Gnrhr, Lhb, and Fshb are not only controlled by developmental transcription factors, but also by epigenetic mechanisms that include the modulation of chromatin structure, and histone modifications

Combination treatment with ionising radiation and gefitinib ('Iressa', ZD1839), an epidermal growth factor receptor (EGFR) inhibitor, significantly inhibits bladder cancer cell growth in vitro and in vivo

Purpose: External beam radiotherapy (EBRT) is the principal bladder-preserving monotherapy for muscle-invasive bladder cancer. Seventy percent of muscle-invasive bladder cancers express epidermal growth factor receptor (EGFR), which is associated with poor prognosis. Ionising radiation (IR) stimulates EGFR causing activation of cytoprotective signalling cascades and thus may be an underlying cause of radioresistance in bladder tumours. Materials and methods: We assessed the ability of IR to activate EGFR in bladder cancer cells and the effect of the anti-EGFR therapy, gefitinib on potential radiation-induced activation. Subsequently we assessed the effect of IR on signalling pathways downstream of EGFR. Finally we assessed the activity of gefitinib as a monotherapy, and in combination with IR, using clonogenic assay in vitro, and a murine model in vivo. Results: IR activated EGFR and gefitinib partially inhibited this activation. Radiation-induced activation of EGFR activated the MAPK and Akt pathways. Gefitinib partially inhibited activation of the MAPK pathway but not the Akt pathway. Treatment with combined gefitinib and IR significantly inhibited bladder cancer cell colony formation more than treatment with gefitinib alone (p = 0.001-0.03). J82 xenograft tumours treated with combined gefitinib and IR showed significantly greater growth inhibition than tumours treated with IR alone (p = 0.04). Conclusions: Combining gefitinib and IR results in significantly greater inhibition of invasive bladder cancer cell colony formation in vitro and significantly greater tumour growth inhibition in vivo. Given the high frequency of EGFR expression by bladder tumours and the low toxicity of gefitinib there is justification to translate this work into a clinical trial.Peer-reviewedPublisher Version1721

The Spatial, Ionization, and Kinematic Conditions of the z=1.39 Damped Ly-alpha Absorber in Q0957+561 A,B

We examined the sizes of the absorption clouds in a z=1.3911 damped Ly-alpha absorber (DLA) in the double image lensed quasar Q0957+561 A,B (separation 135 pc at the absorber redshift). Using HIRES/Keck spectra, we studied the MgII 2796,2803 doublet, FeII multiplet, and MgI 2853 transition in absorption. We defined six "clouds" in the system of sightline A and seven clouds in the system of sightline B. An examination of the N(v) profiles, using the apparent optical depth method, reveals no clear physical connection between the clouds in A and those in B. The observed column density ratios of all clouds is log[N(MgI)/N(FeII)] ~ -2 across the full velocity range in both systems and also spatially (in both sightlines). This is a remarkable uniformity not seen in Lyman limit systems. The uniformity of the cloud properties suggests that the multiple clouds are not part of a "halo". Based upon photoionization modeling, we constrain the ionization parameters in the range -6.2 < log(U) < -5.1, where the range brackets known abundance ratio and dust depletion patterns. The inferred cloud properties are densities of 2 < n_H < 20 cm^-3, and line of sight sizes of 1 < D < 25 pc. The masses of the clouds in system A are 10 < M/M_sun < 1000 and in system B are 1 < M/M_sun < 60 for spherical clouds. For planar clouds, the upper limits are 400 M_sun and 160 M_sun for A and B, respectively. We favor a model of the absorber in which the DLA region itself is a single cloud in thiscomplex, which could be a parcel of gas in a galactic ISM. A spherical cloud of ~10 pc would be limited to one of the sightlines (A) and imply a covering factor less than 0.1 for the DLA complex. We infer that the DLA cloud properties are consistent with those of lower density, cold clouds in the Galactic interstellar medium.Comment: Accepted for publication in the Astrophysical Journal; final versio

North to south asymmetries in the water‐equivalent hydrogen distribution at high latitudes on Mars

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/95381/1/jgre2434.pd