Nicole Wagner

RNA Induction and Inheritance of Epigenetic Cardiac Hypertrophy in the Mouse

Epigenetic regulation shapes normal and pathological mammalian development and physiology. Our previous work showed that Kit RNAs injected into fertilized mouse eggs can produce heritable epigenetic defects, or paramutations, with relevant loss-of-function pigmentation phenotypes, which affect adult phenotypes in multiple succeeding generations of mice. Here, we illustrate the relevance of paramutation to pathophysiology by injecting fertilized mouse eggs with RNAs targeting Cdk9, a key regulator of cardiac growth. Microinjecting fragments of either the coding region or the related microRNA miR-1 led to high levels of expression of homologous RNA, resulting in an epigenetic defect, cardiac hypertrophy, whose efficient hereditary transmission correlated with the presence of miR-1 in the sperm nucleus. In this case, paramutation increased rather than decreased expression of Cdk9. These results highlight the diversity of RNA-mediated epigenetic effects and may provide a paradigm for clinical cases of familial diseases whose inheritance is not fully explained in Mendelian terms.

The Major Podocyte Protein Nephrin Is Transcriptionally Activated by the Wilms’ Tumor Suppressor WT1

NPHS1 encodes the structural protein nephrin, which has a crucial role in the filtration barrier of the glomerular podocyte. Mutations or deregulation of NPHS1 are associated with a variety of renal diseases, including the Finnish type congenital nephrotic syndrome. This study analyzed a potential regulation of nephrin by the Wilms tumor protein, Wt1. Using an inducible U2OS osteosarcoma cell line, it is shown that upon Wt1 induction, endogenous nephrin mRNA becomes highly upregulated. Co-transfection studies demonstrate that Wt1 can activate the nephrin promoter >10-fold. DNase footprinting and mutation analysis identify a Wt1 responsive element in the nephrin promoter, which is required for the binding of Wt1 protein. Mutations or deletion of this Wt1 responsive element completely abolished transactivation of the nephrin promoter by Wt1. Moreover, transgenic analysis demonstrates the requirement of the identified binding site to direct podocyte-specific expression of a reporter gene in transgenic mice, thus confirming the importance of this site for the regulation of nephrin in vivo. Finally, it is shown that nephrin expression is lowest in kidneys of mice that lack specifically the Wt1(-KTS) splice variant, but in comparison with wild-type littermates, it is also reduced in animals with disruption of the Wt1(+KTS) splice variant. Taken together, these data identify nephrin as a direct transcriptional target for Wt1 and underline the importance of Wt1 as a key regulator in podocyte function.

The Wilms' tumor gene Wt1 is required for normal development of the retina.

The Wilms tumor gene Wt1 is known for its important functions during genitourinary and mesothelial formation. Here we show that Wt1 is necessary for neuronal development in the vertebrate retina. Mouse embryos with targeted disruption of Wt1 exhibit remarkably thinner retinas than age‐matched wild‐type animals. A large fraction of retinal ganglion cells is lost by apoptosis, and the growth of optic nerve fibers is severely disturbed. Strikingly, expression of the class IV POU‐domain transcription factor Pou4f2 (formerly Brn‐3b), which is critical for the survival of most retinal ganglion cells, is lost in Wt1−/− retinas. Forced expression of Wt1 in cultured cells causes an up‐regulation of Pou4f2 mRNA. Moreover, the Wt1(−KTS) splice variant can activate a reporter construct carrying 5′‐regulatory sequences of the human POU4F2. The lack of Pou4f2 and the ocular defects in Wt1−/− embryos are rescued by transgenic expression of a 280 kb yeast artificial chromosome carrying the human WT1 gene. Taken together, our findings demonstrate a continuous requirement for Wt1 in normal retina formation with a critical role in Pou4f2‐dependent ganglion cell differentiation.

The complex life of WT1

The Wilms tumour gene, WT1, encodes a zinc-finger transcription factor that is inactivated in a subset of Wilms tumours. Mutation analysis in human patients and genetic experiments in mice have revealed that WT1 has a role much wider than just tumour suppression. Alternative splicing, RNA editing, and the use of alternative translation initiation sites generate a multitude of isoforms, which seem to have overlapping but also distinct functions during embryonic development and the maintenance of organ function. Recently, mouse strains lacking the WT1(-KTS) or WT1(+KTS) splice variants of exon 9 were generated. More severe defects of kidneys and gonads are found in mice lacking the WT1(-KTS) variant. Animals lacking the WT1(+KTS) variant show disturbed podocyte function and male-to-female sex reversal. Alternative splicing of exon 5, however, might not modify WT1 function dramatically. Recently, it was also described that reduction of WT1 levels in the kidney results in glomerulosclerosis and upregulation of WT1 in the heart might contribute to neovascularization after infarction.

The Wilms' tumor suppressor Wt1 is expressed in the coronary vasculature after myocardial infarction.

Expression of the Wilms tumor gene Wt1 in the epicardium is critical for normal heart development. Mouse embryos with inactivated Wt1 gene have extremely thin ventricles, which can result in heart failure and death. Here, we demonstrate that Wt1 can be activated in adult hearts by local ischemia. Wt1 mRNA was increased more than twofold in the left ventricular myocardium of rats between 1 day and 9 wk after infarction. Wt1 expression was localized by means of mRNA in situ hybridization and immunohistochemistry to vascular endothelial and vascular smooth muscle cells in the border zone of the infarcted tissue. A strikingly similar distribution was seen for vascular endothelial growth factor and two different cell proliferation markers in the coronary vessels of the ischemic heart. No Wt1 could be detected in the vasculature of the noninfarcted right ventricles. Wt1 expression in the coronary vessels of the ischemic heart was mimicked by exposure of rats to normobaric hypoxia (8% O2) and 0.1% CO, respectively. These findings demonstrate that Wt1 is expressed in the vasculature of the heart in response to local ischemia and hypoxia. They suggest that Wt1 has a role in the growth of coronary vessels after myocardial infarction.

Rapid Active Zone Remodeling during Synaptic Plasticity

How can synapses change the amount of neurotransmitter released during synaptic plasticity? Although release in general is intensely investigated, its determinants during plasticity are still poorly understood. As a model for plastic strengthening of synaptic release, we here use the well-established presynaptic homeostatic compensation during interference with postsynaptic glutamate receptors at the Drosophila neuromuscular junction. Combining short-term plasticity analysis, cumulative EPSC analysis, fluctuation analysis, and quantal short-term plasticity modeling, we found an increase in the number of release-ready vesicles during presynaptic strengthening. High-resolution light microscopy revealed an increase in the amount of the active zone protein Bruchpilot and an enlargement of the presynaptic cytomatrix structure. Furthermore, these functional and structural alterations of the active zone were not only observed after lifelong but already after minutes of presynaptic strengthening. Our results demonstrate that presynaptic plasticity can induce active zone remodeling, which regulates the number of release-ready vesicles within minutes.

Developmental control of nuclear size and shape by Kugelkern and Kurzkern.

BACKGROUNDnThe shape of a nucleus depends on the nuclear lamina, which is tightly associated with the inner nuclear membrane and on the interaction with the cytoskeleton. However, the mechanism connecting the differentiation state of a cell to the shape changes of its nucleus are not well understood. We investigated this question in early Drosophila embryos, where the nuclear shape changes from spherical to ellipsoidal together with a 2.5-fold increase in nuclear length during cellularization.nnnRESULTSnWe identified two genes, kugelkern and kurzkern, required for nuclear elongation. In kugelkern- and kurzkern-depleted embryos, the nuclei reach only half the length of the wild-type nuclei at the end of cellularization. The reduced nuclear size affects chromocenter formation as marked by Heterochromatin protein 1 and expression of a specific set of genes, including early zygotic genes. kugelkern contains a putative coiled-coil domain in the N-terminal half of the protein, a nuclear localization signal (NLS), and a C-terminal CxxM-motif. The carboxyterminal CxxM motif is required for the targeting of Kugelkern to the inner nuclear membrane, where it colocalizes with lamins. Depending on the farnesylation motif, expression of kugelkern in Drosophila embryos or Xenopus cells induces overproliferation of nuclear membrane.nnnCONCLUSIONSnKugelkern is so far the first nuclear protein, except for lamins, that contains a farnesylation site. Our findings suggest that Kugelkern is a rate-determining factor for nuclear size increase. We propose that association of farnesylated Kugelkern with the inner nuclear membrane induces expansion of nuclear surface area, allowing nuclear growth.

A splice variant of the Wilms' tumour suppressor Wt1 is required for normal development of the olfactory system

Neuronal lineage formation in the developing olfactory epithelium has been extensively studied at the cellular level, but little is known about the genes that control proliferation and differentiation of neuronal progenitor cells. Here, we report that the Wilms tumour zinc-finger protein, Wt1, is required for normal formation of the olfactory epithelium. Wt1 was detected by immunohistochemistry in the developing olfactory epithelium of wild-type embryos between gestational days E9.5 and E18.5. Embryos with complete lack of Wt1 and embryos with selective ablation of the alternatively spliced Wt1(+KTS) isoform both had thinner olfactory epithelia and fewer neuronal progenitor cells than do normal animals. Mash1 and neurogenin 1, two basic helix-loop-helix transcription factors with critical functions during olfactory neuron development, were reduced in the Wt1(+KTS)-/- mutants compared with the wild-type mice. Stable expression of the Wt1(+KTS) isoform, but not of the Wt1(-KTS) variant, upregulated Mash1 mRNA and protein in vitro. The olfactory epithelia of mouse embryos, which lacked the Wt1(-KTS) protein, appeared normal. However, formation of the neural retina was severely impaired in the Wt1(-KTS)-/- mutants. These findings demonstrate that the Wt1(+KTS) protein, which has been proposed to play a role in mRNA processing, acts upstream of Mash1 to promote the development of the olfactory epithelium. Furthermore, neuron formation depends on distinct functions of alternatively spliced Wt1 products in the embryonic retina and the olfactory epithelium.

Peroxisome proliferator-activated receptor β stimulation induces rapid cardiac growth and angiogenesis via direct activation of calcineurin

AIMSnPeroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors. PPARbeta agonists were suggested as potential drugs for the treatment of metabolic syndrome, but effects of PPARbeta activation on cardiac growth and vascularization are unknown. Thus, we investigated the consequences of pharmacological PPARbeta activation on the heart and the underlying molecular mechanisms.nnnMETHODS AND RESULTSnMale C57/Bl6 mice were injected with the specific PPARbeta agonists GW0742 or GW501516, or vehicle. Cardiomyocyte size and vascularisation were determined at different time points. Expression differences were investigated by quantitative reverse transcriptase-polymerase chain reaction and western blotting. In addition, the effects of PPARbeta stimulation were compared with hearts of mice undergoing long-term voluntary exercise or pharmacological PPARalpha activation. Five hours after GW0742 injection, we detected an enhanced angiogenesis compared with vehicle-injected controls. After 24 h, the heart-to-body weight ratios were higher in mice injected with either GW0742 or GW501516 vs. controls. The increased heart size was due to cardiomyocyte enlargement. No signs of pathological cardiac hypertrophy (i.e. apoptosis, fibrosis, or deteriorated cardiac function) could be detected. The effects are mediated via calcineurin A (CnA) activation as: (i) CnA was upregulated, (ii) GW0742 administration or co-transfection of PPARbeta significantly stimulated the activity of the CnA promoter, (iii) PPARbeta protein bound directly to the CnA promoter, (iv) the CnA target genes NFATc3, Hif-1alpha, and Cdk 9 were upregulated in response to PPARbeta stimulation, and (v) the inhibition of CnA activity by cyclosporine A abolished the hypertrophic and angiogenic responses to PPARbeta stimulation.nnnCONCLUSIONnOur data suggest PPARbeta pharmacological activation as a novel approach to increase cardiac vascularization and cardiac muscle mass.

1,25-dihydroxyvitamin D3-induced apoptosis of retinoblastoma cells is associated with reciprocal changes of Bcl-2 and bax.

The active vitamin D metabolite 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) and related substances have previously been tested in tissue culture and animal models of retinoblastoma for their use as anti-tumor drugs. However, despite of the potential therapeutic value, the molecular mechanisms through which 1,25-(OH)(2)D(3) inhibits the growth of retinoblastoma cells are incompletely understood. To elucidate possible signalling pathways for the anti-proliferative action of vitamin D compounds in retinal tumor cells, we analyzed the effect of 1,25-(OH)(2)D(3) and its synthetic analogue KH1060 on the growth of human retinoblastoma-derived Y79 cells. Vitamin D receptor (VDR) mRNA was detected by reverse transcription PCR in Y79 cells and in tissue specimens of human retinoblastoma. VDR transcripts were confirmed at the protein level by strong immunostaining of solid retinal tumors for VDR. Incubation with 1,25-(OH)(2)D(3) and KH1060 (10(-10)-10(-6)moll(-1)) decreased the number of Y79 cells in a timely and dose-dependent manner. Treatment with 1,25-(OH)(2)D(3) (10(-10)moll(-1)) for 24 hr caused cell cycle arrest in the G0/1 phase. Apoptosis of Y79 cells in response to 1,25-(OH)(2)D(3) was demonstrated by the means of TdT-dUTP terminal nick-end labelling (TUNEL), annexin V staining, and detection of DNA fragmentation on agarose gels. 1,25-(OH)(2)D(3)-induced programmed death of Y79 cells was accompanied by a concentration-dependent increase in Bax protein and a reduction in Bcl-2 content. These findings suggest that 1,25-(OH)(2)D(3) inhibits the growth of retinoblastoma cells by causing cell cycle arrest and apoptosis. 1,25-(OH)(2)D(3)-induced programmed death of retinoblastoma cells appears to involve reciprocal changes in Bcl-2 and Bax proteins.