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Frances I Smith

Publications and source records attributed to Frances I Smith.

9 recordsLinked to original sources
PubMed2006

Defects in tangential neuronal migration of pontine nuclei neurons in the Largemyd mouse are associated with stalled migration in the ventrolateral hindbrain.

The LARGE gene encodes a putative glycosyltransferase that is required for normal glycosylation of dystroglycan, and defects in LARGE can cause abnormal neuronal migration in congenital muscular dystrophy (CMD). Previous studies have focused on radial migration, which is disrupted at least in part due to breaks in the basal lamina. Through analysis of precerebellar nuclei development in the Large(myd) mouse hindbrain, we show that tangential migration of a subgroup of hindbrain neurons may also be disrupted. Within the precerebellar nuclei, the pontine nuclei (PN) are severely disrupted, whereas the inferior olive (IO), external cuneate nuclei (ECN) and lateral reticular nuclei (LRN) appear unaffected. Large and dystroglycan are widely expressed in the hindbrain, including in the pontine neurons migrating in the anterior extramural migratory stream (AES). BrdU labeling and immunohistochemical studies suggest normal numbers of neurons begin their journey towards the ventral midline in the AES in the Large(myd) mouse. However, migration stalls and PN neurons fail to reach the midline, surviving as ectopic clusters of cells located under the pial surface dorsally and laterally to where they normally would finish their migration near the ventral midline. Stalling of PN neurons at this location is also observed in other migration disorders in mice. These observations suggest that glycan-dependent dystroglycan interactions are required for PN neurons to correctly respond to signals at this important migrational checkpoint.

Amino Acids↗
PubMed2005

Self-inactivating lentiviruses: versatile vectors for quantitative transduction of cerebellar granule neurons and their progenitors.

Cerebellar granule neurons (CGNs) undergo a well-defined, intrinsic differentiation program that is recapitulated in vitro. Thus, homogeneous cultures of CGNs provide an excellent opportunity to define the mechanisms underlying their development. The ability to alter endogenous gene expression in CGNs on a population-wide basis would greatly facilitate the elucidation of these events. In the present study, we show that self-inactivating lentiviruses efficiently infect both dividing progenitors and post-mitotic CGN cultures in a quantitative manner without altering their cellular properties. The time course for protein expression was biphasic for both types of cultures, with the first peak occurring during the initial infection period. Thus, lentiviruses can express proteins in CGNs both acutely and on a long-term basis to study developmental and other processes continuously over an extended time period. These vectors also infected CGNs in cerebellar slice preparations. In addition, lentiviruses harboring a transgene for the mouse GABA(A) receptor alpha6 subunit promoter recapitulated the differentiation-dependent expression of this gene in CGN cultures. Self-inactivating lentiviruses are extremely versatile vectors that offer important advantages for studies of protein function and gene regulation. The ability to alter protein function on a global scale in CGN cultures permits biochemical assessment of its impact on mRNA and protein populations, as well as on protein--protein and protein--DNA interactions. Further, integrated lentiviruses can be used to study chromatin-dependent promoter regulation and transcription factor interactions in CGNs over time in a facile manner.

Animals↗
PubMed2005

Gene expression profiling of mouse postnatal cerebellar development using oligonucleotide microarrays designed to detect differences in glycoconjugate expression.

Differences in gene expression patterns between adult and postnatal day 7 (P7) mouse cerebellum, at the peak of granule neuron migration, were analyzed by hybridization to the GLYCOv2 glycogene array. This custom designed oligonucleotide array focuses on glycosyl transferases, carbohydrate-binding proteins, proteoglycans and related genes, and 173 genes were identified as being differentially expressed with statistical confidence. Expression levels for 11 of these genes were compared by RT-PCR, and their differential expression between P7 and adult cerebellum confirmed. Within the group of genes showing differential expression, the sialyltransferases (SiaTs) and GalNAc-Ts that were elevated at P7 prefer glycoprotein substrates, whilst the SiaTs and GalNAc-Ts that were elevated in the adult preferentially modify glycolipids, consistent with a role for gangliosides in maintaining neuronal function in the adult. Also within this group, three proteoglycans--versican, bamacan and glypican-2--were elevated at P7, along with growth factor midkine, which is known to bind to multiple types of proteoglycans, and fibroblast growth factor receptor 1, whose activity is known to be influenced by heparan sulfate proteoglycans. Two sulfotransferases that can modify the extent of proteoglycan sulfation were also differentially regulated, and may modify the interaction of a subset of proteoglycans with their binding partners during cerebellar development. Bamacan, glypican-2 and midkine were shown to be expressed in different cell types, and their roles in cerebellar development during granule neuron migration and maturation are discussed.

Aging↗
PubMed2005

Neuronal migration defects in cerebellum of the Largemyd mouse are associated with disruptions in Bergmann glia organization and delayed migration of granule neurons.

The Large gene encodes a putative glycosyltransferase that is required for normal glycosylation of dystroglycan, and defects in either Large or dystroglycan cause abnormal neuronal migration. The mechanism for this effect is not fully understood. This study analyzes the Largemyd mouse cerebellum during postnatal cerebellar development. Large is shown to be expressed most strongly in the Bergmann glial cells and Purkinje cells throughout cerebellar development, which is similar to what is known for dystroglycan expression. Discontinuities of the pial surface of the developing Largemyd mouse cerebellum correlate with disruption of the normal organization of the external granule cell layer and Bergmann glial fibers. At early time points, granule neurons express differentiation markers normally, both temporally and spatially, and show no defects in neurite outgrowth in in vitro assays. However, granule neuron migration is delayed within the external granule and molecular layers, resulting in granule neurons undergoing their intrinsically programmed differentiation in inappropriate locations. Consequently, cells expressing mature granule neuron markers become stranded within these layers. The cause of the less efficient migration is likely due to both physical disruption of the glial-guide scaffolding, as well as to suboptimal neuronal-glial guide interactions during migration.

Animals↗
PubMed2004

Alpha-dystroglycan interactions affect cerebellar granule neuron migration.

The interaction of alpha-dystroglycan (a-DG) with its extracellular binding partners requires glycans attached to its mucin core domain, and defects in the glycosylation of a-DG are associated with both muscular dystrophy and neuronal migration defects. The involvement of a-DG and one of its ligands, agrin, in cerebellar neuronal migration was investigated. Antibodies directed against glycosylated a-DG inhibited granule neuron migration in cerebellar slice cultures. a-DG interactions did not appear to influence neurite outgrowth in cerebellar explant cultures, but enhanced granule neuron binding was observed on cells transfected with a-DG. These results suggest that interactions involving a-DG influence the strength of attachment of granule neurons to the a-DG-expressing Bergmann glial cells that guide granule neuron migration in the cerebellum. Experiments using anti-agrin antibodies suggest that agrin is not involved in these interactions.

Agrin↗
PubMed2004

Developmental expression of receptor for advanced glycation end products (RAGE), amphoterin and sulfoglucuronyl (HNK-1) carbohydrate in mouse cerebellum and their role in neurite outgrowth and cell migration.

Receptor for advanced glycation end products (RAGE) has been proposed as a signal transduction receptor to promote neurite outgrowth and cell migration, by its interaction with a neurite outgrowth promoting protein, Amphoterin. Amphoterin has been shown to interact with sulfoglucuronyl carbohydrate (SGC). The developmental expression of RAGE, Amphoterin and SGC was studied in pre-natal and post-natal mouse cerebellum to establish their cellular and subcellular localization and function. The amount of RAGE in the cerebellum increased with age. RAGE was expressed pre-natally in the external germinal layer and post-natally in the plasma membranes of the granule neurons of the external and internal granule cell layers and in Purkinje cells. Immunocytochemical analysis by high magnification confocal microscopy showed that RAGE was co-expressed with Amphoterin and SGC in the cell surfaces of granule neurons. This co-localization of RAGE, Amphoterin, and SGC was confirmed in isolated and cultured granule neurons and in migrating granule neurons in explant cultures. Anti-RAGE antibodies inhibited neurite outgrowth and cell migration in explant and slice cultures, similar to anti-Amphoterin and anti-SGC antibodies shown previously. The results suggest that RAGE could act as a signaling molecule for neurite outgrowth and cell migration by its interaction with Amphoterin and that of Amphoterin with SGC.

Age Factors↗
PubMed2003

Expression of dystroglycan, fukutin and POMGnT1 during mouse cerebellar development.

Dystroglycan (DG) plays a central role in linking the extracellular matrix to cellular cytoskeletal elements, and is required for proper neuromuscular junction organization and neural cell migration in the CNS. DG interactions with laminin and several other extracellular ligands are mediated through carbohydrates located in a densely glycosylated mucin core domain on alpha-DG. A hallmark of a number of congenital muscular dystrophies is abnormal alpha-DG glycosylation and disordered neuronal migration in both the cerebral cortex and cerebellum. The underlying genetic defects in two such diseases have been localized to the POMGnT1 glycosyltransferase and the putative glycosyltransferase fukutin. We report here the spatial expression pattern of DG together with its putative modifying enzymes during the period of peak neuronal migration in the cerebellum. All three genes are broadly expressed in late embryonic and early postnatal cerebellar neurons, including premigratory granule neurons of the external granule cell layer. Expression of POMGnT1 and fukutin is maintained in neurons of the internal granule cell layer after migration is complete, whereas DG mRNA is largely downregulated. Purkinje cells expressed all three genes throughout development at varying levels, ranging from weak expression of DG to a unique pattern of intense fukutin expression in irregularly spaced cell bodies that do not appear to correlate with known parasagittal stripes. Significantly, immunocytochemical analysis reveals that alpha- and beta-DG proteins are also present on the Bergmann glial scaffolds used by granule cells during early postnatal radial migration, and double-label in situ hybridization confirms that these cells also express POMGnT1 and fukutin. These results suggest that abnormal glycosylation of alpha-DG on glial scaffolds and neurons and their processes could affect interactions with alpha-DG ligands expressed by migrating granule cells, and be a potential mechanism through which neuronal migration is compromised in CMD disease.

Animals↗
PubMed2002

Viral vector-mediated delivery of competing glycosyltransferases modifies epitope expression cell specifically.

The glycoconjugate epitopes 3-fucosyl-N-acetyllactosamine (CD15) and sulfoglucuronylcarbohydrate (SGC) mediate cell adhesion events in several systems, and are regulated both spatially and temporally during cerebellar development. In cotransfection studies using COS-1 cells, competition between glycosyltransferases that utilize a common precursor involved in the final synthetic steps of these epitopes, can modulate epitope expression. For example, cotransfection of rat alpha1,3-fucosyltransferase IV (Fuc-TIV) and either rat glucuronic acid transferase P (GlcAT) or pig alpha1,3-galactosyltransferase (GalT) resulted in the dominance of either SGC or GalalphaGal epitope expression, respectively, with blockage of CD15 epitope expression. Viral vectors expressing these glycosyltransferases were used to determine whether competition plays a role in establishing epitope dominance in cerebellar cells, and whether overexpression of competing glycosyltransferases could be used to block epitope expression. Infection of cerebellar astrocytes with viral vectors expressing either Fuc-TIV, or Fuc-TIX, caused dramatic increases in CD15 expression in the presence of continued endogenous SGC epitope expression. Likewise, viral transduction with GalT resulted in GalalphaGal expression without affecting endogenous CD15 or SGC expression. Thus, competition between these enzymes does not appear to play a role in establishing epitope expression in astrocytes, and transduction of these enzymes does not provide a method of blocking the expression of endogenous epitopes. In contrast to what was observed for astrocytes, infection with viral vectors expressing either Fuc-T, GlcAT, or GalT did not result in significant expression of the relevant epitopes (CD15, SGC or GalalphaGal, respectively) on granule neurons. These results suggest a different complement of precursors are present in granule neurons and astrocytes, presumably due to the presence of different complements of glycosyltransferases in these cells.

Animals↗