Jenny M. Gunnersen

Growth and migration markers of rat C6 glioma cells identified by serial analysis of gene expression

Tumors derived from rat C6 cell implants into rat brain exhibit similar morphological characteristics and degree of vascularization to human glioblastomas. To establish a molecular basis for C6 gliosarcoma malignancy, we have constructed a molecular profile of the most abundantly expressed genes, using serial analysis of gene expression (SAGE). Sequence tags (1168) representing 738 individual transcripts were collected and tag‐to‐gene mapping was carried out using the UniGene data set for rat. Differentially expressed C6 transcripts were identified by comparison of tags collected for C6 cells with a similar number (1002) of tags from a rat primary astrocyte library. Genes found to be expressed at increased levels in C6 cells are associated with cell surface interactions, migration, or metastasis formation and proliferation. These include the receptor for hyaluronan‐mediated motility (RHAMM), S‐100 related protein 42A, galectin I, preproenkephalin, osteopontin, autocrine motility factor, α‐tubulin, ad1 antigen, and cofilin. In addition, a tag with no database match probably representing a previously uncharacterized transcript was differentially expressed in C6 cells. Transcripts showing reduced expression in C6 cells relative to astrocytes included the extracellular matrix glycoprotein osteonectin/SPARC (secreted protein, acidic, rich in cysteine), actin‐binding proteins thymosins β‐4 and β‐10, the cysteine protease inhibitor cystatin C, the actin‐gelling protein SM22/transgelin, and ferritin‐H. SAGE results were confirmed by Northern blot for all transcripts tested, reaffirming the value of the SAGE technique for expression profiling in cancer biology. GLIA 32:146–154, 2000.

Expression of the relaxin gene in rat tissues.

The peptide hormone relaxin is produced at high levels in the corpus luteum of the rat ovary during pregnancy. The biological effects of the hormone include remodelling of collagen in target tissues such as the cervix, and inhibition of uterine contractility. In addition, many paracrine actions for the hormone have been proposed, however sites of relaxin production outside the ovary have not been well characterized. We have investigated relaxin gene expression in a range of rat tissues using the techniques of reverse transcription/polymerase chain reaction (RT/PCR), RNase protection and immunohistochemistry. Relaxin mRNA was detected by RT/PCR in brain, uterus, prostate gland, pancreas and kidney, with other tissues giving weak signals. Relaxin gene expression in brain was detected by RNase protection, and relaxin-like immunoreactivity was observed in the arcuate nucleus of the hypothalamus of rat brain. This characterization of sites of relaxin gene expression provides further evidence for proposed paracrine actions of relaxin which may be important in non-pregnant and male rats in addition to the pregnant female.

Sez-6 Proteins Affect Dendritic Arborization Patterns and Excitability of Cortical Pyramidal Neurons

Development of appropriate dendritic arbors is crucial for neuronal information transfer. We show, using seizure-related gene 6 (sez-6) null mutant mice, that Sez-6 is required for normal dendritic arborization of cortical neurons. Deep-layer pyramidal neurons in the somatosensory cortex of sez-6 null mice exhibit an excess of short dendrites, and cultured cortical neurons lacking Sez-6 display excessive neurite branching. Overexpression of individual Sez-6 isoforms in knockout neurons reveals opposing actions of membrane-bound and secreted Sez-6 proteins, with membrane-bound Sez-6 exerting an antibranching effect under both basal and depolarizing conditions. Layer V pyramidal neurons in knockout brain slices show reduced excitatory postsynaptic responses and a reduced dendritic spine density, reflected by diminished punctate staining for postsynaptic density 95 (PSD-95). In behavioral tests, the sez-6 null mice display specific exploratory, motor, and cognitive deficits. In conclusion, cell-surface protein complexes involving Sez-6 help to sculpt the dendritic arbor, in turn enhancing synaptic connectivity.

Automated analysis of neurite branching in cultured cortical neurons using HCA-Vision

Manual neuron tracing is a very labor‐intensive task. In the drug screening context, the sheer number of images to process means that this approach is unrealistic. Moreover, the lack of reproducibility, objectivity, and auditing capability of manual tracing is limiting even in the context of smaller studies. We have developed fast, sensitive, and reliable algorithms for the purpose of detecting and analyzing neurites in cell cultures, and we have integrated them in software called HCA‐Vision, suitable for the research environment. We validate the software on images of cortical neurons by comparing results obtained using HCA‐Vision with those obtained using an established semi‐automated tracing solution (NeuronJ). The effect of the Sez‐6 deletion was characterized in detail. Sez‐6 null neurons exhibited a significant increase in neurite branching, although the neurite field area was unchanged due to a reduction in mean branch length. HCA‐Vision delivered considerable speed benefits and reliable traces.

Expression of human relaxin genes: characterization of a novel alternatively-spliced human relaxin mRNA species.

Relaxin is a two-chain peptide hormone encoded by two non-allelic genes in humans and great apes, and by a single gene in all other species studied. We have characterized the expression of the human relaxin genes (H1 and H2) in placenta, decidua, prostate and ovary by reverse-transcription/polymerase chain reaction (RT/PCR). H2 relaxin mRNA was detected in the ovary, term placenta, decidua, and prostate gland. In contrast, H1 gene expression was detected only in the prostate gland. In addition to the relaxin PCR product of the predicted size (486 bp), a larger relaxin-specific product (587 bp) was detected in both H1 and H2 amplifications and in amplifications of chimpanzee relaxin from placenta and corpus luteum. Sequencing of human and chimpanzee PCR products, and human relaxin genomic clones, revealed that the larger product arises from an alternatively-spliced relaxin mRNA species incorporating an extra exon. This is the first evidence that the structure of the human and chimpanzee relaxin genes differ from that of other characterized relaxin genes, such as pig and rat. The novel peptide arising from this alternate message would be identical to prorelaxin in the B-chain and part of the C-peptide (extending to the position of the intron) but would differ from prorelaxin in the carboxy-terminal domain. Observation of a similar mRNA species in the chimpanzee suggests that this conserved relaxin-like peptide may have a significant biological role.

Layer positioning of late-born cortical interneurons is dependent on Reelin but not p35 signaling.

We tested the response of interneurons to the absence of Reelin signaling or p35 in the mouse neocortex. We provide three independent strands of evidence to demonstrate that layering of late-born (but not early-born) interneurons is regulated by Reelin signaling. First, early-born and late-born interneurons behaved differently in mice lacking Reelin or disabled 1 (Dab1). Early-born interneurons showed layer inversion, whereas late-born interneurons did not demonstrate layer inversion but were randomly distributed across the cortex. Second, in p35 mutant brains (in which Reelin signaling is intact), late-born interneurons are appropriately positioned in the upper layers despite the malpositioning of all other cortical neurons in these mice. Third, transplanted late-born interneuron precursors (wild type) into Dab1−/− cortices showed appropriate upper layer segregation. Together, these results indicate that, in the absence of Reelin signaling, late-born interneurons fail to laminate properly, and this is restored in an environment in which Reelin signaling is intact. These studies suggest different mechanisms for the stratification of cortical interneurons. Whereas the early-born interneurons appear to be associated with projection neuron layering, late-born interneurons rely on Reelin signaling for their correct lamination.

MicroSAGE is highly representative and reproducible but reveals major differences in gene expression among samples obtained from similar tissues

BackgroundSerial analysis of gene expression using small amounts of starting material (microSAGE) has not yet been conclusively shown to be representative, reproducible or accurate.ResultsWe show that microSAGE is highly representative, reproducible and accurate, but that pronounced differences in gene expression are seen between tissue samples taken from different individuals.ConclusionsMicroSAGE is a reliable method of comprehensively profiling differences in gene expression among samples, but care should be taken in generalizing results obtained from libraries constructed from tissue obtained from different individuals and/or processed or stored differently.

Nedd4-WW Domain-Binding Protein 5 (Ndfip1) Is Associated with Neuronal Survival after Acute Cortical Brain Injury

Understanding the transcriptional response to neuronal injury after trauma is a necessary prelude to formulation of therapeutic strategies. We used Serial Analysis of Gene Expression (SAGE) to identify 50,000 sequence tags representing 18,000 expressed genes in the cortex 2 h after traumatic brain injury (TBI). A similar tag library was obtained from sham-operated cortex. The SAGE data were validated on biological replicates using quantitative real-time-PCR on multiple samples at 2, 6, 12, and 24 h after TBI. This analysis revealed that the vast majority of genes showed a downward trend in their pattern of expression over 24 h. This was confirmed for a subset of genes using in situ hybridization and immunocytochemistry on brain sections. Of the overexpressed genes in the trauma library, Nedd4-WW (neural precursor cell expressed, developmentally downregulated) domain-binding protein 5 (N4WBP5) (also known as Ndfip1) is strongly expressed in surviving neurons around the site of injury. Overexpression of N4WBP5 in cultured cortical neurons increased the number of surviving neurons after gene transfection and growth factor starvation compared with control transfections. These results identify N4WBP5 as a neuroprotective protein and, based on its known interaction with the ubiquitin ligase Nedd4, would suggest protein ubiquitination as a possible survival strategy in neuronal injury.

Global analysis of gene expression patterns in developing mouse neocortex using serial analysis of gene expression.

Molecular inventories of the developing mouse neocortex before and after birth were generated using the global gene expression profiling tool serial analysis of gene expression (SAGE). Libraries were generated from embryonic day 15 and postnatal day 1 mouse neocortex and more than 40,000 tags were collected (20,211 and 22,001 tags, representing 11,706 and 12,402 transcripts, respectively). Comparison of the two libraries resulted in the identification of 321 transcripts that were differentially expressed (P < 0.05). Differential expression was independently verified for selected genes by Northern blotting, and in situ hybridization revealed spatial expression patterns in the neocortex. Differentially expressed transcripts included genes known to be important in neocortical development (e.g., brain factor 1, neuroD2, and Id2), genes not previously associated with neocortical development (such as brahma-related gene 1, receptor for activated C-kinase I, hypermethylated in cancer 2, and Evi9), and genes of unknown identity or function.

Place- and time-dependent expression of mouse sFRP-1 during development of the cerebral neocortex.

Wnts are a family of secreted proteins involved in multiple developmental mechanisms during nervous system development, including cell proliferation, cell migration, axon guidance and specification of cell positional information. We report here the expression of sFRP-1 mRNA, encoding a putative inhibitor of Wnt, in the developing mouse neocortex during the entire period when neurons for the neocortex are born. We show that sFRP-1 mRNA expression is spatially restricted to the proliferative zones during the period, when neurons are known to be generated in large numbers for the enlarging cortical plate.