A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain

RNA-interference-driven loss of function in specific tissues in vivo should permit analysis of gene function in temporally and spatially defined contexts. However, delivery of efficient short hairpin RNA (shRNA) to target tissues in vivo remains problematic. Here, we demonstrate that efficiency of polyethylenimine (PEI)-delivered shRNA depends on the regulatory sequences used, both in vivo and in vitro. When tested in vivo, silencing of a luciferase target gene by shRNA produced from a hybrid construct composed of the CMV enhancer/promoter placed immediately upstream of an H1 promoter (50%) exceeds that obtained with the H1 promoter alone (20%). In contrast, in NIH 3T3 cells, the H1 promoter was more efficient than the hybrid construct (75 versus 60% inhibition of target gene expression, respectively). To test CMV-H1 shRNA efficiency against an endogenous gene in vivo, we used shRNA against thyroid hormone receptor α1 (TRα1). When vectorized in the mouse brain, the hybrid construct strongly derepressed CyclinD1-luciferase reporter gene expression, CyclinD1 being a negatively regulated thyroid hormone target gene. We conclude that promoter choice affects shRNA efficiency distinctly in different in vitro and in vivo situations and that a hybrid CMV-H1 construct is optimal for shRNA delivery in the mouse brain

Satellite and Ground Remote Sensing Techniques to Trace the Hidden Growth of a Lava Flow Field: The 2014-15 Effusive Eruption at Fogo Volcano (Cape Verde)

Fogo volcano erupted in 2014–2015 producing an extensive lava flow field in the summit caldera that destroyed two villages, Portela and Bangaeira. The eruption started with powerful explosive activity, lava fountains, and a substantial ash column accompanying the opening of an eruptive fissure. Lava flows spreading from the base of the eruptive fissure produced three arterial lava flows. By a week after the start of the eruption, a master lava tube had already developed within the eruptive fissure and along the arterial flow. In this paper, we analyze the emplacement processes based on observations carried out directly on the lava flow field, remote sensing measurements carried out with a thermal camera, SO2 fluxes, and satellite images, to unravel the key factors leading to the development of lava tubes. These were responsible for the rapid expansion of lava for the ~7.9 km length of the flow field, as well as the destruction of the Portela and Bangaeira villages. The key factors leading to the development of tubes were the low topography and the steady magma supply rate along the arterial lava flow. Comparing time-averaged discharge rates (TADR) obtained from satellite and Supply Rate (SR) derived from SO2 flux data, we estimate the amount and timing of the lava flow field endogenous growth, with the aim of developing a tool that could be used for hazard assessment and risk mitigation at this and other volcanoes.This research received no external funding.Published11156V. Pericolosità vulcanica e contributi alla stima del rischioJCR Journa

Role of Foxl2 in uterine maturation and function

International audienceFoxl2 codes for a forkhead/HNF3 transcription factor essential for follicular maturation and maintenance of ovarian identity. FOXL2 mutations are associated with Blepharophimosis, Ptosis and Epicanthus inversus Syndrome (BPES) characterized by eyelid malformations (types I and II) and premature ovarian insufficiency (type I). We show that Foxl2 is not only expressed by the ovary, but also by other components of the mouse female reproductive tract, including the uterus, the cervix and the oviduct. In the uterus, Foxl2 expression is first observed in the neonatal mesenchyme and, during uterine maturation, persists in the stroma and in the deep inner myometrial layer (IML). In the adult, Foxl2 is expressed in the differentiated stromal layer, but no longer in the myometrium. Conditional deletion of Foxl2 in the postnatal (PN) uterus using Progesterone Receptor-cre (Pgr(cre/+)) mice results in infertility. During PN uterine maturation Pgr(cre/+); Foxl2(flox/flox) mice present a severely reduced thickness of the stroma layer and an hypertrophic, disorganized IML. In adult Pgr(cre/+); Foxl2(flox/flox) mice a supplementary muscular layer is present at the stroma/myometrium border and vascular smooth muscle cells fail to form a coherent layer around uterine arteries. Wnt signalling pathways play a central role in uterine maturation; in Pgr(cre/+); Foxl2(flox/flox) mice, Wnt genes are deregulated suggesting that Foxl2 acts through these signals. In humans, thickening of the IML (also called "junctional zone") is associated with reduced fertility, endometriosis and adenomyosis. Our data suggest that Foxl2 has a crucial role in PN uterine maturation and could help to understand sub-fertility predisposition in women

Role of Foxl2 in uterine maturation and function

International audienceFoxl2 codes for a forkhead/HNF3 transcription factor essential for follicular maturation and maintenance of ovarian identity. FOXL2 mutations are associated with Blepharophimosis, Ptosis and Epicanthus inversus Syndrome (BPES) characterized by eyelid malformations (types I and II) and premature ovarian insufficiency (type I). We show that Foxl2 is not only expressed by the ovary, but also by other components of the mouse female reproductive tract, including the uterus, the cervix and the oviduct. In the uterus, Foxl2 expression is first observed in the neonatal mesenchyme and, during uterine maturation, persists in the stroma and in the deep inner myometrial layer (IML). In the adult, Foxl2 is expressed in the differentiated stromal layer, but no longer in the myometrium. Conditional deletion of Foxl2 in the postnatal (PN) uterus using Progesterone Receptor-cre (Pgr(cre/+)) mice results in infertility. During PN uterine maturation Pgr(cre/+); Foxl2(flox/flox) mice present a severely reduced thickness of the stroma layer and an hypertrophic, disorganized IML. In adult Pgr(cre/+); Foxl2(flox/flox) mice a supplementary muscular layer is present at the stroma/myometrium border and vascular smooth muscle cells fail to form a coherent layer around uterine arteries. Wnt signalling pathways play a central role in uterine maturation; in Pgr(cre/+); Foxl2(flox/flox) mice, Wnt genes are deregulated suggesting that Foxl2 acts through these signals. In humans, thickening of the IML (also called "junctional zone") is associated with reduced fertility, endometriosis and adenomyosis. Our data suggest that Foxl2 has a crucial role in PN uterine maturation and could help to understand sub-fertility predisposition in women

A CMV-H1 hybrid construct driving shLuc provides enhanced inhibition of a co-transfected target gene in the newborn (a and b) and in the adult (c) mouse brain

<p><b>Copyright information:</b></p><p>Taken from "A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain"</p><p></p><p>Nucleic Acids Research 2007;35(9):e65-e65.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1888798.</p><p>© 2007 The Author(s)</p> () Dose dependence of CMV-H1-shLuc efficiency. The inhibition efficiency of CMV-H1-shLuc was tested at different doses ranging from 0.1 to 0.4 µg/µl. After co-transfection of pGL2-CMV and pRL-CMV along with 0.4 µg/µl of CMV-H1-shLuc (i.e. 0.8 µg/hemisphere), we observed up to 50% inhibition of the targeted luciferase expression. This level of inhibition was obtained at 48 h post-transfection. () Time course efficiency of CMV-H1-shLuc. Significant inhibition of the target gene with 0.4 µg/µl of CMV-H1-shLuc was seen at all times tested. The maximal level of inhibition (50%) was seen at 50 h post-transfection. () In the adult brain, H1-shLuc provided no inhibition of PP:RL ratio (grey bars) compared to controls (black bars). CMV-H1-shLuc leads to 25% inhibition of the target gene at 72 h post-transfection (white bars) and up to 112 h post-transfection (data not shown). Means ± SEM are shown. NS = ‘not significant’; * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001.  = 10 injected hemispheres per group

Dlx5 and Dlx6 control uterine adenogenesis during post-natal maturation: Possible consequences for endometriosis

Dlx5 and Dlx6 are two closely associated homeobox genes which code for transcription factors involved in the control of steroidogenesis and reproduction. Inactivation of Dlx5/6 in the mouse results in a Leydig cell defect in the male and in ovarian insufficiency in the female. DLX5/6 are also strongly expressed by the human endometrium but their function in the uterus is unknown. The involvement of DLX5/6 in human uterine pathology is suggested by their strong downregulation in endometriotic lesions and upregulation in endometrio\uefd adenocarcinomas. We first show that Dlx5/6 expression begins in M\ufcllerian ducts epithelia and persists then in the uterine luminal and glandular epithelia throughout post-natal maturation and in the adult. We then use a new mouse model in which Dlx5 and Dlx6 can be simultaneously inactivated in the endometrium using a Pgrcre/+ allele. Post-natal inactivation of Dlx5/6 in the uterus results in sterility without any obvious ovarian involvement. The uteri of Pgrcre/+; Dlx5/6flox/flox mice present very few uterine glands and numerous abnormally large and branched invaginations of the uterine lumen. In Dlx5/6 mutant uteri, the expression of genes involved in gland formation (Foxa2) and in epithelial remodelling during implantation (Msx1) is significantly reduced. Furthermore, we show that DLX5 is highly expressed in human endometrial glandular epithelium and that its expression is affected in endometriosis. We conclude that Dlx5 and Dlx6 expression determines uterine architecture and adenogenesis and is needed for implantation. Given their importance for female reproduction, DLX5 and DLX6 must be regarded as interesting targets for future clinical research

Dlx5 and Dlx6 expression in GABAergic neurons controls behavior, metabolism, healthy aging and lifespan.

International audienceDlx5 and Dlx6 encode two homeobox transcription factors expressed by developing and mature GABAergic interneurons. During development Dlx5/6 are important for the differentiation of Parvalbumin (Pvalb)-expressing neurons. Perinatal lethality of homozygous mice in which Dlx5/6 have been constitutively deleted has, so far, hindered the study of the function of these genes in adult neurons. We first show that Dlx5 and Dlx6 are expressed by all subclasses of adult cortical GABAergic neurons. Then we analyse VgatΔDlx5-6 mice in which Dlx5 and Dlx6 are simultaneously inactivated in all GABAergic interneurons. VgatΔDlx5-6 mice present a behavioral pattern suggesting reduction of anxiety and obsessivecompulsive activities. They rapidly access and spend more time in the central region of an open field, bury few marbles in the marble burying test and show little interest in nest building. Male and female 20-month-old VgatΔDlx5-6 animals have the same size as their normal littermates, but present a 25% body weight reduction associated with a marked decline in white and brown adipose tissue. Remarkably, both VgatΔDlx5-6/+ and VgatΔDlx5-6 mice present a 33% longer median survival than their control littermates. Hallmarks of biological aging such as motility, adipose deposition and coat conditions are improved in mutant animals. Our data imply that GABAergic interneurons can regulate mammalian healthspan and lifespan through Dlx5/6-dependent mechanisms. Understanding these regulations can be an entry point to unravel the processes through which the brain affects body homeostasis and, ultimately, longevity and healthy aging

Probing the origin of matching functional jaws: roles of Dlx5/6 in cranial neural crest cells

Abstract Gnathostome jaws derive from the first pharyngeal arch (PA1), a complex structure constituted by Neural Crest Cells (NCCs), mesodermal, ectodermal and endodermal cells. Here, to determine the regionalized morphogenetic impact of Dlx5/6 expression, we specifically target their inactivation or overexpression to NCCs. NCC-specific Dlx5/6 inactivation (NCC ∆Dlx5/6 ) generates severely hypomorphic lower jaws that present typical maxillary traits. Therefore, differently from Dlx5/6 null-embryos, the upper and the lower jaws of NCC ∆Dlx5/6 mice present a different size. Reciprocally, forced Dlx5 expression in maxillary NCCs provokes the appearance of distinct mandibular characters in the upper jaw. We conclude that: (1) Dlx5/6 activation in NCCs invariably determines lower jaw identity; (2) the morphogenetic processes that generate functional matching jaws depend on the harmonization of Dlx5/6 expression in NCCs and in distinct ectodermal territories. The co-evolution of synergistic opposing jaws requires the coordination of distinct regulatory pathways involving the same transcription factors in distant embryonic territories