Developmental neurobiology. Recitative and aria.
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Glia Maturation Factor↗
Biomedical subjects
G Lemke
Publications and source records attributed to G Lemke.
The molecular genetics of myelination: an update.
Recent molecular genetic studies have provided new insights into the structure and function of 2 of the major integral membrane proteins of myelin--the proteolipid protein (PLP) and protein zero (P0)--and have uncovered a third such protein--PMP22/gas3. The rumpshaker mouse has been shown to carry a point mutation in the PLP gene that uncouples a deleterious effect on CNS myelin assembly, which these mice exhibit, from oligodendrocyte degeneration and cell death, which they do not. The developmental importance of the P0 protein in PNS myelination has been dramatically demonstrated by the analysis of loss-of-function mutations engineered through the expression of antisense RNA and through the insertional inactivation of the P0 gene by homologous recombination in embryonic stem cells and the generation of P0-deficient mice. The cloned promoter of the P0 gene has been shown to drive quantitative, Schwann cell-specific expression of heterologous genes in transgenic mice. The PMP22/gas3 gene, previously cloned from fibroblast cell lines, has been found to encode an axonally regulated Schwann cell protein that is assembled into PNS myelin. Importantly, this gene appears to be the target of mutations that result in the Trembler alleles in mice, and in Charcot-Marie-Tooth disease Type 1a, the most common inherited peripheral neuropathy in humans.
Animals↗
Single cell analysis of the expression of a nuclear protein, SCIP, by fluorescent immunohistochemistry visualized with confocal microscopy.
A widely applicable method for the accurate quantification or semiquantification of macromolecules at the level of individual cells is described and validated; this is a method which may considerably facilitate the study of many biological processes. This method relies on measuring fluorescent emission in immunocytochemically labelled cells with a confocal microscope. Emission is related quantitatively to the level of the fluorophore by the combination of an analysis of the polarization of the fluorescent emission and fluorophore rationing methods. The method was applied to the study of the expression of the suppressed cyclic AMP-induced POU protein (SCIP) transcription factor in glial cells of the central nervous system. In particular, the method allowed the study of transcription factor expression in defined cells present in heterogeneous cultures and in cell types which cannot be isolated in sufficient numbers for biochemical analysis using conventional techniques.
Animals↗
Repression of the myelin P0 gene by the POU transcription factor SCIP.
SCIP is a POU domain transcription factor expressed by Schwann cells, the myelin-forming glial cells of the peripheral nervous system. In this study, we investigate SCIP regulation of the gene encoding P0, the major structural protein of peripheral myelin. We find that SCIP represses transcription of this gene through the joint action of the SCIP POU domain and an amino terminal domain that acts cell specifically. Maximal repression is DNA-binding-dependent, and analysis of the P0 promoter reveals the presence of multiple SCIP binding sites. Surprisingly, none of these sites in their native positions dramatically affect P0 promoter activity or its repression by SCIP, although they mediate repression when moved closer to the P0 transcription start site. We propose that repression occurs through a quenching mechanism mediated by the SCIP POU and amino terminal domains acting in concert with other nuclear proteins, including a Schwann cell-specific adapter.
Animals↗
Transcriptional regulation of the development of neurons and glia.
The past year has seen significant progress in the analysis of transcriptional regulation as it relates to neural development. Highlights include the identification and analysis of new homeobox genes that delimit developmental boundaries in the vertebrate forebrain, the study of upstream regulators of homeobox genes, the analysis of Pax genes that may contribute to specification of the vertebrate dorso-ventral neuraxis, and the functional analysis of transcription factors that are likely to specify particular neural cell types in both vertebrate and invertebrate nervous systems.
Animals↗
Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons.
We have used homologous recombination in embryonic stem cells to generate mice carrying a mutation in the gene encoding P0, an immunoglobulin-related recognition molecule and the major protein of peripheral nervous system myelin. These mice are deficient in normal motor coordination and exhibit tremors and occasional convulsions. Axons in their peripheral nerves are severely hypomyelinated and a subset of myelin-like figures and axons degenerate. The mutation leads to an abnormal regulation of some, but not all, molecules involved in myelination. These results demonstrate that P0 is essential for the normal spiraling, compaction, and maintenance of the peripheral myelin sheath and the continued integrity of associated axons. They further suggest that this protein conveys a signal that regulates Schwann cell gene expression.
Animals↗
P0 promoter directs expression of reporter and toxin genes to Schwann cells of transgenic mice.
We generated transgenic mice that specifically express foreign genes in myelinating Schwann cells. A 1.1 kb segment of 5' flanking sequence from the rat P0 gene was used to drive expression of the genes encoding human growth hormone (hGH) and bacterial diphtheria toxin A chain (DT-A). The P0-hGH mice expressed hGH in myelinating Schwann cells, but not in nonmyelinating Schwann cells, the central nervous system, or any other tissue assayed. This expression was activated on a developmental schedule comparable to that of endogenous myelin gene expression. One line of P0-DT-A mice developed a generalized hypomyelinating peripheral neuropathy, with Schwann cell deficiency apparent in newborn animals. Peripheral nerves from adult mice of this line displayed morphological alterations ranging from completely denuded axons to myelinated Schwann cells undergoing degeneration, although occasional Schwann cells were able to form apparently normal myelin sheaths. Pronounced secondary changes, including proliferation and retraction of processes, occurred in the nonmyelinating Schwann cells of these P0-DT-A mice.
Age Factors↗
Down-regulation of the POU transcription factor SCIP is an early event in oligodendrocyte differentiation in vitro.
The POU-domain transcription factor SCIP (also known as Tst-1) has been implicated in the development of Schwann cells, the myelinating cells of the peripheral nervous system (PNS). We have investigated the possibility that SCIP also might play a role in the development of oligodendrocytes, the myelinating cells of the central nervous system (CNS). We purified oligodendrocyte precursors (O-2A progenitors) by immunoselection and cultured them in the presence of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), which together keep O-2A progenitors proliferating and prevent oligodendrocyte differentiation. Under these culture conditions, O-2A progenitors expressed high levels of SCIP mRNA and protein, and did not express myelin-specific genes. When oligodendrocyte differentiation was initiated by withdrawing the growth factors, SCIP mRNA was rapidly down-regulated, followed by a decline in SCIP protein and the sequential activation of myelin-specific genes. Rapid down-regulation of SCIP mRNA required continued protein synthesis. In O-2A progenitors that were cultured in the presence of PDGF alone, SCIP expression declined to an intermediate level, and low levels of the myelin gene products were induced. Thus, the level of SCIP expression in O-2A progenitors is inversely related to the level of myelin gene expression, suggesting that SCIP may be involved in the developmental switch from proliferation to differentiation in the oligodendrocyte lineage. When O-2A progenitors are cultured in the presence of 10% fetal calf serum, they differentiate into type-2 astrocytes rather than oligodendrocytes. SCIP mRNA was also down-regulated in type-2 astrocytes, which do not express myelin genes, so down-regulation of SCIP seems to be more closely linked to the cessation of cell proliferation per se than the expression of a particular differentiated phenotype.
Animals↗
Notch2: a second mammalian Notch gene.
Notch is a cell surface receptor that mediates a wide variety of cellular interactions that specify cell fate during Drosophila development. Recently, homologs of Drosophila Notch have been isolated from Xenopus, human and rat, and the expression patterns of these vertebrate proteins suggest that they may be functionally analogous to their Drosophila counterpart. We have now identified a second rat gene that exhibits substantial nucleic and amino acid sequence identity to Drosophila Notch. This gene, designated Notch2, encodes a protein that contains all the structural motifs characteristic of a Notch protein. Thus, mammals differ from Drosophila in having more than one Notch gene. Northern and in situ hybridisation analyses in the developing and adult rat identify distinct spatial and temporal patterns of expression for Notch1 and Notch2, indicating that these genes are not redundant. These results suggest that the great diversity of cell-fate decisions regulated by Notch in Drosophila may be further expanded in vertebrates by the activation of distinct Notch proteins.
Amino Acid Sequence↗
Axon-regulated expression of a Schwann cell transcript that is homologous to a 'growth arrest-specific' gene.
We have isolated a 1.8 kb cDNA (pCD25) clone that encodes a transcript that is differentially expressed during nerve regeneration. Nucleotide sequence comparison indicates 89.6% homology with the recently identified murine 'growth arrest-specific' gene gas3. The open reading frame of the CD25 transcript predicts a 17 kDa protein with four putative transmembrane regions. Steady-state levels of the CD25 mRNA are very much higher in sciatic nerve than in other tissues, and expression in sciatic nerve is confined to Schwann cells. Following nerve injury, the transcript levels rapidly declined in nerve segments distal to the site of lesion, but recovered upon nerve regeneration. In contrast, in distal stumps of permanently transected nerves, the mRNA level remained very low. Substantial amounts of the mRNA could be reinduced only upon anastomosis of these interrupted nerve stumps. Re-induction of the mRNA followed the elongation of regenerating axons through the distal nerve segment. Our data indicate that axons regulate expression of the CD25 mRNA in Schwann cells, and suggest that the CD25 protein functions during Schwann cell growth and differentiation.
Amino Acid Sequence↗
An extended family of protein-tyrosine kinase genes differentially expressed in the vertebrate nervous system.
We have used PCR to identify 13 novel protein-tyrosine kinase genes (tyro-1 to -13), six of which (tyro-1 to -6) are preferentially expressed in the developing vertebrate nervous system. The tyro-2 and tyro-9 genes encode kinase domains that exhibit strong amino acid sequence similarity to the equivalent regions of the receptors for EGF and FGF, respectively, and may encode novel receptors for these or related polypeptide ligands. The tyro-1 to -6 genes are all expressed during central nervous system neurogenesis and exhibit distinct and highly regionalized patterns of expression in the adult brain. Together with recent studies in invertebrates, these data are consistent with the hypothesis that protein-tyrosine kinases play a central role in neural development.
Amino Acid Sequence↗
Expression and activity of the transcription factor SCIP during glial differentiation and myelination.
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Aging↗
The gene encoding the transcription factor SCIP has features of an expressed retroposon.
SCIP is a POU domain transcription factor expressed by glial progenitor cells in the peripheral and central nervous systems (dividing Schwann cells and O-2A cells, respectively), where it appears to act as a repressor of myelin-specific genes. We have isolated genomic clones encoding the rat SCIP gene. Comparison of the structure of these clones with genomic Southern blots and SCIP cDNAs demonstrates that SCIP is encoded in a single-copy, intronless gene that has the general features of an expressed retroposon. This gene contributes to an extended CpG island. It is transcribed to produce a 3.1-kb mRNA that encodes a 451-amino-acid protein with a predicted molecular mass of 45 kDa. Immunopurified SCIP antibodies specifically recognize a nuclear protein of this size in cultured proliferating Schwann cells, and gel shift analyses demonstrate that this protein is the predominant octamer-binding protein in these cells.
Animals↗
A homolog of Drosophila Notch expressed during mammalian development.
Drosophila Notch and the related Caenorhabditis elegans proteins lin-12 and glp-1 function as mediators of local cell-cell interactions required for cell-fate decisions during invertebrate development. To investigate the possibility that similar proteins play determinative roles during mammalian development, we isolated cDNA clones encoding rat Notch. The deduced amino acid sequence of this protein contains 36 epidermal growth factor (EGF)-like repeats, and is remarkably similar in both its extracellular and cytoplasmic domains to the sequence of Xenopus Xotch and Drosophila Notch. In the developing central nervous system, in situ hybridisation analyses revealed that Notch transcripts were dramatically restricted to the ventricular proliferative zones of embryonic neuroepithelia. Notch was also strongly expressed during development of non-neural tissues, such as hair follicles and tooth buds, whose correct differentiation requires epithelial-mesenchymal interactions. These data support the hypothesis that Notch plays an essential role in mammalian development and pattern formation that closely parallels its role in the development of invertebrates.
Amino Acid Sequence↗
Growth factors and transcription factors in oligodendrocyte development.
O-2A progenitor cells, the precursors of oligodendrocytes in the central nervous system (CNS), probably originate in the subventricular germinal zones of the developing CNS, and subsequently migrate away from there to populate the rest of the CNS with oligodendrocytes. We are trying to understand how the O-2A progenitor cells interact with their changing environment as they migrate, and how this influences each stage of their development into mature, myelinating oligodendrocytes. In this article we summarize evidence that platelet-derived growth factor (PDGF) is important for stimulating O-2A progenitor cell proliferation in vivo, and describe our efforts to map the distribution of PDGF and its receptors in the developing rat CNS by in situ hybridization and immunohistochemistry. These studies suggest that, in the CNS, PDGF alpha-receptor subunits may be restricted to O-2A lineage cells that have started to migrate away from the subventricular zones towards their final destinations. Many neurons express the A and/or B chains of PDGF, and astrocytes express the A chain, but it is not yet clear which of these cell types might be the major source of PDGF for O-2A lineage cells in vivo. O-2A progenitor cells can be purified and maintained in a proliferating state in vitro by culturing in the presence of PDGF and bFGF. Under these conditions, the POU transcription factor SCIP/Tst-1 is expressed at a high level; when oligodendrocyte differentiation is initiated by withdrawing the growth factors, SCIP/Tst-1 mRNA is rapidly down-regulated, followed by a decline in SCIP/Tst-1 protein and sequential activation of myelin-specific genes.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Induction of the myelin proteolipid protein (PLP) gene in C6 glioblastoma cells: functional analysis of the PLP promotor.
The terminal differentiation of postmitotic oligodendrocytes is marked by the induction of myelin-specific genes. In this report, we demonstrate that culture conditions that induce oligodendrocyte differentiation of glial progenitor cells also induce differentiation of C6 glioblastoma cells, as monitored by activated transcription of the gene encoding proteolipid protein (PLP), the major myelin protein of the CNS. When assayed by transfections of hybrid reporter plasmids, the transcriptional control region of the PLP gene is preferentially active in differentiated C6 cells and contains both positive and negative cis-regulatory elements. In general, functional identification of these elements is well correlated with the binding sites of glial nuclear proteins, as visualized by the presence of DNase I-protected footprints. A sequence within one positive cis-regulatory element of the PLP gene is conserved in the control regions of three other myelin-specific genes, suggesting that their coordinate transcription may involve a common regulatory mechanism.
Animals↗
Expression and activity of the POU transcription factor SCIP.
POU proteins have been shown to transcriptionally active cell-specific genes and to participate in the determination of cell fate. It is therefore thought that these proteins function in development through the stable activation of genes that define specific developmental pathways. Evidence is provided here for an alternative mode of action. The primary structure of SCIP, a POU protein expressed by developing Schwann cells of the peripheral nervous system, was deduced and SCIP activity was studied. Both in normal development and in response to nerve transection, SCIP expression was transiently activated only during the period of rapid cell division that separates the premyelinating and myelinating phases of Schwann cell differentiation. In cotransfection assays, SCIP acted as a transcriptional repressor of myelin-specific genes.
Amino Acid Sequence↗