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.

SCIP: a glial POU domain gene regulated by cyclic AMP.

We have isolated cDNA clones encoding SCIP, a POU domain gene expressed by myelin-forming glial of the central and peripheral nervous systems. In purified Schwann cells cultured in the absence of neurons, expression of SCIP is suppressed. This suppression is relieved by cAMP, and induction of SCIP mRNA by this second messenger precedes cAMP induction of myelin-specific genes. Similarly, SCIP expression in vivo precedes full expression of myelin-specific genes in developing oligodendrocytes and Schwann cells. The sequence of the SCIP POU domain is identical to that of Tst-1, a recently identified member of a family of POU domain genes expressed by restricted subsets of neurons. Our results demonstrate that SCIP is also expressed by myelin-forming glia and suggest that it plays a central role in the progressive determination of these cells and their commitment to myelination.

Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-Lhx2 pathway.

The organizing centers and molecules that pattern the cerebral cortex have been elusive. Here we show that cortical patterning involves regulation of the Lhx2 homeobox gene by the roof plate. Roof plate ablation results in reduced cortical size and Lhx2 expression defects that implicate roof plate signals in the bimodal regulation of Lhx2 in vivo. Bimodal Lhx2 regulation can be recapitulated in explants using two roof plate-derived signaling molecules, Bmp4 and Bmp2. Loss of Lhx2 function results in profound losses of cortical progenitors and neurons, but Lhx2 mutants continue to generate cortical neurons from dorsal sources that may include the roof plate region itself. These findings provide evidence for the roof plate as an organizing center of the developing cortex and for a roof plate-Lhx2 pathway in cortical patterning.

Mechanisms of cerebral cortical patterning in mice and humans.

All the higher mental and cognitive functions unique to humans depend on the neocortex ('new' cortex, referring to its relatively recent appearance in evolution), which is divided into discrete areas that subserve distinct functions, such as language, movement and sensation. With a few notable exceptions, all neocortical areas have six layers of neurons and a remarkably similar thickness and overall cell density, despite subtle differences in their cellular architecture. Furthermore, all neocortical areas are formed over roughly the same time period during development and provide little hint at early developmental stages of the rich functional diversity that becomes apparent as development comes to an end. How these areas are formed has long fascinated developmental neuroscientists, because the formation of new cortical areas, with the attendant appearance of new cortical functions, is what must have driven the evolution of mammalian behavior.

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.

Pituicytoma: diagnostic features on selective carotid angiography and MR imaging.

We report a case of pituicytoma, a rare primary tumor of the neurohypophysis. A 64-year-old man presented with progressive visual complaints, bitemporal hemianopsia, and headache. Imaging studies revealed distinctive features of a mass lesion that thickened the pituitary stalk with a bilobed protrusion extending into the hypothalamus. Angiography demonstrated tumor vascular supply from the superior hypophyseal arteries representing the diencephalic branches of the internal carotid arteries. We discuss the imaging and pathology of this unusual tumor.

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.

Cell-specific action and mutable structure of a transcription factor effector domain.

POU proteins are cell-specific transcription factors whose specificity of action has been attributed to protein-DNA and protein-protein interactions mediated by their DNA-binding (POU) domains. Here we report that transcriptional activation by SCIP, a POU protein expressed by developing Schwann cells, is dependent on an amino-terminal effector domain and that this domain mediates cell-specific transactivation in the complete absence of the POU domain. When fused to a heterologous DNA-binding domain, this SCIP domain is a potent transactivator in Schwann cells but is inactive in three heterologous cell types. The primary structure of the SCIP amino-terminal domain is novel but contains a polymorphic string of alanine residues similar to those found in several other transcription factors. Although previously hypothesized to be important for transcription factor activity, we find that the SCIP string is functionally irrelevant. We propose that homopolymers of alanine, and certain other amino acids, do not represent a motif required for transcription factor function but instead reflect regions of unstable DNA related to those associated with four recently characterized human genetic disorders.

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.

Biochemical, biomolecular and cellular sensing in microfluidics using flow induced admittance spectra.

We present flow induced admittance spectra for electrolytes, cell culture media, different sizes of DNA solutions and neural cells using flow induced admittance spectra in a microfluidics device. The device comprises of a PDMS channel aligned with a pair of channel electrodes fabricated on glass. The peak of the flow induced admittance spectra and frequency at which the peak occurs are the key parameters used for the characterization of sensing. The response of this sensor is a function of the conductivity and dielectrivity of the effective solution. The flow induced admittances of the particles studied are corrected with their media. This sensing will be a primary component of an electrical based cytometer.

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.

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)