The genetic hierarchy that controls myelination of peripheral nerves by Schwann cells includes the POU domain Oct-6/Scip/Tst-1 and the zinc-finger Krox-20/Egr2 transcription factors. These pivotal transcription factors act to control the onset of myelination during development and tissue regeneration in adults following damage. In this report we demonstrate the involvement of a third transcription factor, the POU domain factor Brn-2. We show that Schwann cells express Brn-2 in a developmental profile similar to that of Oct-6 and that Brn-2 gene activation does not depend on Oct-6. Overexpression of Brn-2 in Oct-6-deficient Schwann cells, under control of the Oct-6 Schwann cell enhancer (SCE), results in partial rescue of the developmental delay phenotype, whereas compound disruption of both Brn-2 and Oct-6 results in a much more severe phenotype. Together these data strongly indicate that Brn-2 function largely overlaps with that of Oct-6 in driving the transition from promyelinating to myelinating Schwann cells. The high conduction velocity of nerve fibers is a hallmark of the nervous system of higher vertebrates and depends on structural and molecular specializations that are elaborated during development. These specializations occur through intimate and continued interactions between the neuron and its associated glial cells and result in the elaboration by glial cells of myelin, the important membranous structure that ensheaths and insulates axons (Arroyo and Scherer 2000; Fields and StevensGraham 2002;Mirsky et al. 2002). Two glial cell types produce myelin: the oligodendrocyte in the central nervous system (CNS) and the Schwann cell in the peripheral nervous system (PNS). Although very similarly organized, the molecular composition of CNS and PNS myelin differs significantly, and oligodendrocytes and Schwann cells have adopted different, but overlapping, sets of transcriptional regulators to coordinate myelogenesis (Hudson 2001; Topilko and Meijer 2001). These differences reflect their distinct embryonic origins. Whereas oligodendrocytes originate from the neuroepithelial precursors that line the lumen of the spinal cord and ventricles of the brain, Schwann cells derive mainly from the neural crest, a transient embryonic stem (ES) cell population that generates a wide variety of cell types including sensory and autonomic neurons and melanocytes (Le Douarin and Kalcheim 1999;Richardson 2001). Schwann cell precursors populate the early outgrowing nerve bundles, where they proliferate and segregate individual and groups of fibers until the number of Schwann cells and fibers is eventually matched. During the first few days of postnatal development, many Schwann cells establish a 1:1 relationship with axons, cease to proliferate, and initiate myelin formation such that by the end of the first postnatal week of development, all myelin-competent axons are actively being myelinated. Schwann cells that remain associated with groups of lower-caliber fibers will segregate these fibers in cytoplasmic cuffs without mye...

The POU transcription factor Oct-6, also known as SCIP or Tst-1, has been implicated as a major transcriptional regulator in Schwann cell differentiation. Microscopic and immunochemical analysis of sciatic nerves of Oct-6(-/-) mice at different stages of postnatal development reveals a delay in Schwann cell differentiation, with a transient arrest at the promyelination stage. Thus, Oct-6 appears to be required for the transition of promyelin cells to myelinating cells. Once these cells progress past this point, Oct-6 is no longer required, and myelination occurs normally.

The translocation (6;9) is associated with a specific subtype of acute myeloid leukemia (AML). Previously, it was found that breakpoints on chromosome 9 are clustered in one of the introns of a large gene named Cain (can). cDNA probes derived from the 3' part of can detect an aberrant, leukemia-specific 5.5-kb transcript in bone marrow cells from t(6;9) AML patients. cDNA cloning of this mRNA revealed that it is a fusion of sequences encoded on chromosome 6 and 3' can. A novel gene on chromosome 6 which was named dek was isolated. In dek the t(6;9) breakpoints also occur in one intron. As a result the dek-can fusion gene, present Defined karyotypic aberrations are associated with specific subtypes of leukemia. Detailed molecular characterization of these aberrations may identify genes involved in leukemogenesis and in the precise regulation of proliferation and differentiation in the hematopoietic system. Translocations are the best-studied chromosomal abnormalities. As the result of a translocation, the function or activity of oncogenes located at or near the translocation breakpoint is altered. In myeloid leukemia three translocation breakpoints have been cloned and analyzed at the molecular level.The two best studied, t(9;22) in chronic myeloid leukemia (27, 43) and t(15;17) in acute promyelocytic leukemia (2,8,12), result in the formation of chimeric genes that encode fusion proteins. In chronic myeloid leukemia this is a BCR-ABL protein that has an enhanced tyrosine kinase activity (34, 49) directly responsible for its in vivo tumorigenic potential (14,25). In acute promyelocytic leukemia a PMLRARa fusion protein that represents an altered transcription factor (16, 33) is found.The third translocation is the t(6;9) (p23;q34), found in a specific subtype of acute myeloid leukemia (AML) (1,39,41). This leukemia is characterized by a poor prognosis, affects young adults, and is classified mostly as M2 or M4 and rarely as Ml (according to the French-American-British classification of AML). A region on chromosome 9 situated 360 kb downstream of the c-abl gene was cloned and analyzed. It was found that breakpoints were clustered in a region of 8 kb in five patients, four with t(6;9) AML and one with acute undifferentiated leukemia (AUL) (47). Through cDNA cloning this region could be identified as one of the introns of a large gene (>100 kb) encoding a 7-kb transcript. This intron was named icb-9; the intron containing the breakpoints on chromosome 9 and situated in the middle of * Corresponding author. a gene named Cain (can). The 3' part of can is translocated to the 6p-chromosome, and only 3' can probes detect an additional, leukemia-specific 5.5-kb transcript in bone marrow cells from t(6;9) AML patients. No additional transcripts were detected with 5' can probes. The breakpoint region on chromosome 6p23 was isolated from a genomic XEMBL3 library constructed of bone marrow DNA from one of the t(6;9) patients. An area of 40 kb of chromosome 6 DNA was cloned in overlapping phages. Southern blot analysis sh...

Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Crerecombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation.

Williams syndrome is a neurodevelopmental disorder caused by the hemizygous deletion of 1.6 Mb on human chromosome 7q11.23. This region comprises the gene CYLN2, encoding CLIP-115, a microtubule-binding protein of 115 kD. Using a gene-targeting approach, we provide evidence that mice with haploinsufficiency for Cyln2 have features reminiscent of Williams syndrome, including mild growth deficiency, brain abnormalities, hippocampal dysfunction and particular deficits in motor coordination. Absence of CLIP-115 also leads to increased levels of CLIP-170 (a closely related cytoplasmic linker protein) and dynactin at the tips of growing microtubules. This protein redistribution may affect dynein motor regulation and, together with the loss of CLIP-115-specific functions, underlie neurological alterations in Williams syndrome.

Epilepsy is one of the most common and intractable brain disorders. Mutations in the human gene LGI1, encoding a neuronal secreted protein, cause autosomal dominant lateral temporal lobe epilepsy (ADLTE). However, the pathogenic mechanisms of LGI1 mutations remain unclear. We classified 22 reported LGI1 missense mutations as either secretion defective or secretion competent, and we generated and analyzed two mouse models of ADLTE encoding mutant proteins representative of the two groups. The secretion-defective LGI1(E383A) protein was recognized by the ER quality-control machinery and prematurely degraded, whereas the secretable LGI1(S473L) protein abnormally dimerized and was selectively defective in binding to one of its receptors, ADAM22. Both mutations caused a loss of function, compromising intracellular trafficking or ligand activity of LGI1 and converging on reduced synaptic LGI1-ADAM22 interaction. A chemical corrector, 4-phenylbutyrate (4PBA), restored LGI1(E383A) folding and binding to ADAM22 and ameliorated the increased seizure susceptibility of the LGI1(E383A) model mice. This study establishes LGI1-related epilepsy as a conformational disease and suggests new therapeutic options for human epilepsy.

Peripheral nerve development results from multiple cellular interactions between axons, Schwann cells and the surrounding mesenchymal tissue. The delayed axonal sorting and hypomyelination throughout the peripheral nervous system of claw paw (clp) mutant mice suggest that the clp gene product is critical for these interactions. Here we identify the clp mutation as a 225-bp insertion in the Lgi4 gene. Lgi4 encodes a secreted and glycosylated leucine-rich repeat protein and is expressed in Schwann cells. The clp mutation affects Lgi4 mRNA splicing, resulting in a mutant protein that is retained in the cell. Additionally, siRNA-mediated downregulation of Lgi4 in wild-type neuron-Schwann cell cocultures inhibits myelination, whereas exogenous Lgi4 restores myelination in clp/clp cultures. Thus, the abnormalities observed in clp mice are attributable to the loss of Lgi4 function, and they identify Lgi4 as a new component of Schwann cell signaling pathway(s) that controls axon segregation and myelin formation.

The remarkable high affinity (Kd approximately 10(-15) M) of avidin/streptavidin for biotin has been extensively exploited in purification methodologies. Recently a small peptide sequence (Avi-tag) has been defined that can be specifically and efficiently biotinylated by the bacterial BirA biotin ligase. Fusion of this small peptide sequence to a protein of interest and co-expression with the BirA gene in mammalian cells allowed purification of the biotinylated protein together with its associated proteins and other molecules. Ideally, one would like to apply these technologies to purify tagged proteins directly from mouse tissues. To make this approach feasible for a large variety of proteins we developed a mouse strain that expresses the BirA gene ubiquitously by inserting it in the ROSA26 locus. We demonstrate that the BirA protein is indeed expressed in all tissues tested. In order to demonstrate functionality we show that it biotinylates the transgene-encoded Avi-tagged Gata1 and Oct6 transcription factors in erythroid cells of the foetal liver and Schwann cells of the peripheral nerve respectively. Therefore, this mouse can be crossed to any transgenic mouse to obtain efficient biotinylation of an Avi-tagged protein for the purpose of protein (complex) purification.