A potassium channel mutation in weaver mice implicates membrane excitability in granule cell differentiation.

Early events in neuronal differentiation are generally considered to be regulated by factors independent of alterations in membrane permeability. Weaver mice harbour a mutation that blocks neuronal differentiation just after cessation of cell division, prior to cell migration and synaptogenesis. Cerebellar granule cells in homozygous weaver mice fail to differentiate, either because intrinsic cues are absent or because the granule cells are unable to respond to those cues. We now report that weaver mice have a missense mutation in a gene encoding a G-protein coupled inward rectifier potassium channel. The mutation alters the putative ion-permeable, pore-forming domain of the protein, suggesting that granule cell differentiation is regulated by changes in membrane permeability.

Atrophins' emerging roles in development and neurodegenerative disease.

The Atrophins are a widely expressed family of transcriptional co-regulators found in all metazoans. Atrophin1 was first identified as a neurodegenerative disease gene whereas Atrophin2 was identified based on homology. Phylogenetic studies indicate that the primordial Atrophin was an Atrophin2 type of gene and Atrophin2 has critical functions in normal mouse embryonic development whereas Atrophin1 is dispensable. Atrophins can interact with a wide range of proteins including membrane receptors, nuclear hormone receptors and other DNA binding transcription factors and can shuttle between the cytoplasm and the nucleus. In the nucleus, Atrophins can act as either co-repressors or co-activators and taken together this suggests that they are intermediaries in transcriptional responses to a diverse array of exogenous signals. Despite progress in understanding the normal role of Atrophins, the mechanism whereby mutations in Atrophin1 cause neurodegeneration has remained enigmatic, although most studies have focused on the idea that neurodegeneration is related to inappropriate transcriptional repression.

Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel.

The neurotransmitter serotonin (5HT) activates a variety of second messenger signaling systems and through them indirectly regulates the function of ion channels. Serotonin also activates ion channels directly, suggesting that it may also mediate rapid, excitatory responses. A complementary DNA clone containing the coding sequence of one of these rapidly responding channels, a 5HT3 subtype of the serotonin receptor, has been isolated by screening a neuroblastoma expression library for functional expression of serotonin-gated currents in Xenopus oocytes. The predicted protein product has many of the features shared by other members of the ligand-gated ion channel family. The pharmacological and electrophysiological characteristics of the cloned receptor are largely consistent with the properties of native 5HT3 receptors. Messenger RNA encoding this receptor is found in the brain, spinal cord, and heart. This receptor defines a new class of excitatory ligand-gated channels.

Nonselective and G betagamma-insensitive weaver K+ channels.

Homozygous weaver mice are profoundly ataxic because of the loss of granule cell neurons during cerebellar development. This granule cell loss appears to be caused by a genetic defect in the pore region (Gly156-->Ser) of the heterotrimeric guanine nucleotide-binding protein (G protein)-gated inwardly rectifying potassium (K+) channel subunit (GIRK2). A related subunit, GIRK1, associates with GIRK2 to constitute a neuronal G protein-gated inward rectifier K+ channel. The weaver allele of the GIRK2 subunit (wvGIRK2) caused loss of K+ selectivity when expressed either as wvGIRK2 homomultimers or as GIRK1-wvGIRK2 heteromultimers. The mutation also let to loss of sensitivity to G protein betagamma dimers. Expression of wvGIRK2 subunits let to increased cell death, presumably as a result of basal nonselective channel opening.

The tumour-suppressor Scribble dictates cell polarity during directed epithelial migration: regulation of Rho GTPase recruitment to the leading edge.

Altered expression of human Scribble is associated with invasive epithelial cancers, however, its role in tumour development remains unclear. Mutations in Drosophila Scribble result in loss of polarity, overproliferation and 3D-tumourous overgrowth of epithelial cells. Using complementation studies in Drosophila we recently demonstrated that expression of human Scribble can also regulate polarity and restrict tissue overgrowth. Here, we have undertaken a detailed study of human Scribble function in the polarized mammary cell line, MCF10A. We show that although Scribble does not seem to be required for apical-basal polarity or proliferation control in MCF10A cells, Scribble is essential for the control of polarity associated with directed epithelial cell migration. Scribble-depleted MCF10A cells show defective in vitro wound closure and chemotactic movement. The cells at the wound edge fail to polarize, show reduced lamellipodia formation and impaired recruitment of Cdc42 and Rac1 to the leading edge. Furthermore, we show that this function is relevant in vivo as Scribble mutant mice show defective epidermal wound healing. This data identifies an essential role for mammalian Scribble in the regulation of the polarity specifically involved in directed epithelial migration.

A genetic screen for behavioral mutations that perturb dopaminergic homeostasis in mice.

Disruption of dopaminergic (DA) systems is thought to play a central role in the addictive process and in the pathophysiology of schizophrenia. Although inheritance plays an important role in the predisposition to these disorders, the genetic basis of this is not well understood. To provide additional insight, we have performed a modifier screen in mice designed to identify mutations that perturb DA homeostasis. With a genetic background sensitized by a mutation in the dopamine transporter (DAT), we used random chemical mutagenesis and screened for mutant mice with locomotor abnormalities. Four mutant lines were identified with quantitatively elevated levels of locomotor activity. Mapping of mutations in these lines identified two loci that alter activity only when dopamine levels are elevated by a DAT mutation and thus would only have been uncovered by this type of approach. One of these quantitative trait loci behaves as an enhancer of DA neurotransmission, whereas the other may act as a suppressor. In addition, we also identified three loci which are not dependent on the sensitized background but which also contribute to the overall locomotor phenotype.

Localization of the scaffolding protein RACK1 in the developing and adult mouse brain.

RACK1 is a multifunctional scaffolding protein known to be involved in the regulation of various signaling cascades in the central nervous system (CNS). In order to gain insight into the neurological functions of RACK1, we examined the expression of RACK1 mRNA and protein during gestation and in the adult mouse brain. Several expression patterns were observed. At embryonic day 11.5 (E11.5), RACK1 is expressed in a high-dorsal to low-ventral gradient throughout the brain. At E13.5, RACK1 is most abundant in the telencephalon. In the developing cortical primordium, RACK1 protein is expressed in a high-rostromidline to low-caudolateral gradient that appears to be regulated post-transcriptionally. At E18.5, RACK1 is expressed most abundantly in layers 1-4 of the cortex, striatum, hippocampus, dentate gyrus and specific thalamic nuclei. In the adult mouse, RACK1 is ubiquitously expressed in neuronal perikarya in most brain regions, with relatively higher levels in hippocampus, olfactory bulb, cortex and cerebellum. Subcellular staining was detected mainly in the cell bodies and extending into dendrites, whereas RACK1 was not present significantly in axonal fibers or nuclei. We also determined brain regions in which RACK1 interacts with one of its binding partners, the betaII isoform of protein kinase C (betaIIPKC). We found that betaIIPKC had a much more restricted expression pattern than RACK1 and overlapped with the scaffolding protein only in certain regions, including the CA1 area of the hippocampus, cerebellum and striatum. Our results suggest an important role for RACK1 during CNS development and support multiple functions of the protein in the adult brain.

Cloning, characterization, and chromosomal localization of rec1.3, a member of the G-protein-coupled receptor family highly expressed in brain.

During a project to identify G-protein-coupled receptors (GPCR) expressed within taste buds, we have isolated a novel receptor-like sequence. The full length sequence of this receptor (rec1.3) has been obtained in both cow and mouse. Rec1.3 bears little sequence similarity to any GPCR whose ligand is known: the closest identity (33%) is to the orphan receptor edg-1. In cow, rec1.3 is expressed most prominently in the brain, with moderate expression in testis and tongue; in the mouse the expression is more widespread. No specific binding for a range of ligands was detected when the mouse coding sequence was expressed in eukaryotic cells. In situ hybridization showed that rec1.3 is widely expressed throughout the mouse brain and is highly expressed in localized regions of the hindbrain, midbrain and hypothalamus. The rec1.3 gene was localized to the centromeric region of chromosome 4 in mouse, a region associated with neonatal seizures.

Urinary incontinence. Basic types and their management in older patients.

Current guidelines for evaluation and management of urinary incontinence in older patients advise thorough history taking, physical examination, and limited laboratory workup. Most patients with urinary incontinence can be treated successfully with behavior modification, muscle exercises, and medications. Surgery should be reserved for patients who are not relieved by conservative therapy or who have obvious anatomical abnormalities amenable to repair.

Benign hereditary nephritis.

The diagnosis, prognosis, and heredity of glomerulonephritis, in 207 members of one family over five generations was determined. No evidence of significant renal insufficiency or renal associated mortality was found. A history of urinary tract infections was obtained from 58 percent of those with hematuria. Several of those with nephritis also had high-frequency hearing losses. Two females with histories of urinary infections had elevated levels of serum creatinine. Many cases will probably be discovered if families are tested when a patient presents with benign recurrent hematuria with or without red blood cell casts. There may not be an obvious history of kidney disease or hematuria. Hereditary nephritis is not always a progressive renal disease.

Racial variation in the incidence of ovarian cancer in the United States.

The incidence of ovarian cancer was determined in four US populations of heterogeneous racial-ethnic composition. Japanese, Chinese, Hispano, and black women had rates of epithelial tumors that were 19-42% lower than those of white women. Though these differences were due primarily to lower rates of serious and papillary tumors, all but Chinese women also had a decreased incidence of mucinous tumors, and Hispano and black women had low rates of endometrioid-clear cell malignancy. The incidence of nonepithelial ovarian tumors in the four populations showed little relationship to race.

Developmental expression of aquaporin-3 in zebrafish embryos (Danio rerio).

Fish embryos have never been successfully cryopreserved because of the low permeability of cryoprotectants into the yolk. Recently, we used aquaporin-3 fused with a green fluorescent protein (AQP3GFP) to modify the zebrafish embryo, and demonstrated that the pores functioned physiologically. This increased the water and cryoprotectant permeability of the membranes. We have continued our work on AQP3-modified embryos and here we report their developmental expression of AQP3, the success of various culture media on their survival and development, and their reproductive success. The AQP3GFP expression begins within 30 m after the mRNA AQP3GFP injection into the yolk of the 1- to 4-cell embryo. This expression is distributed in the membranes throughout the blastoderm and the yolk syncytial layer within 24 h. It diminishes after 96 h. We found no difference in the survival or normal development of embryos from AQP3GFP or wild-type adults. Additionally, zebrafish embryos did not require special culture medium to survive after AQP3GFP modification. In fact, they survived best in embryo medium (ca. 40 mOsm). Embryos reared entirely in embryo medium had a higher percent survival and a higher percent normal development than those exposed to a high osmolality sucrose culture medium (ca. 330 mOsm). The mechanism whereby these embryos can maintain their internal osmolality in a hypoosmotic solution with water channels in their membranes is unknown.

oto is a homeotic locus with a role in anteroposterior development that is partially redundant with Lim1.

Genetic control of mammalian head development involves mechanisms that are shared with trunk development as well as mechanisms that are independent. For example, mutations in the nodal gene disrupt axis formation and head development while mutations in the Otx2 or Lim1 genes block head development without disrupting development of the trunk. We show here that the oto mutation on mouse chromosome 1 defines a locus with a critical role in anterior development. The oto mutation disrupts development of the telencephalic and optic vesicles, the pharyngeal endoderm and the first branchial arch. Also, oto embryos have dose-dependent, posterior homeotic transformations throughout the axial skeleton. To further dissect the role of the oto locus in head development, we crossed mice carrying oto and Lim1 mutations. Interactions between the two mutations indicate that the role of oto in the regulation of head development is partially redundant with that of Lim1. The phenotype of oto embryos points to an early and critical role for oto in the development of forebrain subregions. Transformations of the vertebrae in oto embryos reveal a Lim1-independent role in the establishment of positional information in the trunk.

FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse.

The FKBP-12-rapamycin associated protein (FRAP, also known as mTOR and RAFT-1) is a member of the phosphoinositide kinase related kinase family. FRAP has serine/threonine kinase activity and mediates the cellular response to mitogens through signaling to p70s6 kinase (p70(s6k)) and 4E-BP1, resulting in an increase in translation of subsets of cellular mRNAs. Translational up-regulation is blocked by inactivation of FRAP signaling by rapamycin, resulting in G(1) cell cycle arrest. Rapamycin is used as an immunosuppressant for kidney transplants and is currently under investigation as an antiproliferative agent in tumors because of its ability to block FRAP activity. Although the role of FRAP has been extensively studied in vitro, characterization of mammalian FRAP function in vivo has been limited to the immune system and tumor models. Here we report the identification of a loss-of-function mutation in the mouse FRAP gene, which illustrates a requirement for FRAP activity in embryonic development. Our studies also determined that rapamycin treatment of the early embryo results in a phenotype indistinguishable from the FRAP mutant, demonstrating that rapamycin has teratogenic activity.