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We have generated previously transgenic rats that overexpress peripheral myelin protein 22 (PMP22) in Schwann cells. In the nerves of these animals, Schwann cells have segregated with axons to the normal 1:1 ratio but remain arrested at the promyelinating stage, apparently unable to elaborate myelin sheaths. We have examined gene expression of these dysmyelinating Schwann cells using semiquantitative reverse transcription-PCR and immunofluorescence analysis. Unexpectedly, Schwann cell differentiation appears to proceed normally at the molecular level when monitored by the expression of mRNAs encoding major structural proteins of myelin. Furthermore, an aberrant coexpression of early and late Schwann cell markers was observed. PMP22 itself acquires complex glycosylation, suggesting that trafficking of the myelin protein through the endoplasmic reticulum is not significantly impaired. We suggest that PMP22, when overexpressed, accumulates in a late Golgi–cell membrane compartment and uncouples myelin assembly from the underlying program of Schwann cell differentiation.

NR3B is a modulatory subunit of the NMDA receptor, abundantly expressed in both cranial and spinal somatic motoneurons and at lower levels in other regions of the brain as well. Recently, we found the human NR3B gene (GRIN3B) to be highly genetically heterogeneous, and that approximately 10% of the normal European-American population lacks NR3B due to homozygous occurrence of a null allele in the gene. Therefore, it is especially important to understand the phenotypic consequences of the genetic loss of NR3B in both humans and animal models. We here provide results of behavioral analysis of mice genetically lacking NR3B, which is an ideal animal model due to homogeneity in genetic and environmental background. The NR3B(-/-) mice are viable and fertile. Consistent with the expression of NR3B in somatic motoneurons, the NR3B(-/-) mice showed a moderate but significant impairment in motor learning or coordination, and decreased activity in their home cages. Remarkably, the NR3B(-/-) mice showed a highly increased social interaction with their familiar cage mates in their home cage but moderately increased anxiety-like behaviour and decreased social interaction in a novel environment, consistent with the inhibitory role of NR3B on the functions of NMDA receptors. This work is the first reporting of the functional significance of NR3B in vivo and may give insight into the contribution of genetic variability of NR3B in the phenotypic heterogeneity among human population.

The embryonal carcinoma cell line P19 responds to treatment with retinoid acid by differentiation into neuronal cell types [2]. Using radioactively labeled cDNA derived from differentiating P19 cells we screened an adult mouse brain cDNA library and isolated a gene named shyc for selective hybridizing clone. The encoded protein did not reveal homology to any known protein. We used in situ hybridization on mouse embryonic and adult brain sections to study shyc expression. The developing and embryonic nervous system showed the most prominent hybridization signals. In the adult brain the olfactory pathway was marked by shyc expression.

Tetra-amelia is a rare human genetic disorder characterized by complete absence of all four limbs and other anomalies. We studied a consanguineous family with four affected fetuses displaying autosomal recessive tetra-amelia and craniofacial and urogenital defects. By homozygosity mapping, the disease locus was assigned to chromosome 17q21, with a maximum multipoint LOD score of 2.9 at markers D17S931, D17S1785, D17SS1827, and D17S1868. Further fine mapping defined a critical interval of approximately 8.9 Mb between D17S1299 and D17S797. We identified a homozygous nonsense mutation (Q83X) in the WNT3 gene in affected fetuses of the family. WNT3, a human homologue of the Drosophila wingless gene, encodes a member of the WNT family known to play key roles in embryonic development. The Q83X mutation truncates WNT3 at its amino terminus, suggesting that loss of function is the most likely cause of the disorder. Our findings contrast with the observation of early lethality in mice homozygous for null alleles of Wnt3. To our knowledge, this is the first report of a mutation in a WNT gene associated with a Mendelian disorder. The identification of a WNT3 mutation in tetra-amelia indicates that WNT3 is required at the earliest stages of human limb formation and for craniofacial and urogenital development.

In the mammalian nervous system, the formation of myelin byglial cells enables a rapid saltatory impulse conduction. Myelina-tion depends on the coordinated expression of myelin-specificstructural proteins, based on interactions of axons with oligoden-drocytes in the central nervous system (CNS) and Schwann cells inthe peripheral nervous system (PNS). Mice with defects in myelinassembly comprise a large and genetically heterogeneous group ofspontaneous mutants (Nave 1994), which also serve as models forhuman neurological diseases (Snipes and Suter 1995). Several my-elin-deficient mice carry defects in genes yet unidentified.The murine autosomal mutation claw paw (genotype clp/clp)produces abnormalities of limb posture associated with a myelina-tion disorder of the peripheral nervous system, but not the centralnervous system (Henry et al. 1991). Peripheral nerve myelinationis greatly delayed, and the failure to myelinate results in the per-sistence of promyelin Schwann cell/axon units, involving predomi-nantly small-caliber fibers. The clinical abnormalities, which areobvious within 2 days after birth, include abnormally flexed andextended joints, most obvious in the forelimbs. In human, congen-ital joint contractures (arthrogryposis) may be a presenting featureof congenital hypomyelinating neuropathy (Boylan et al. 1992)and have been associated with mutations in the gene for the majorperipheral myelin protein P0 (Warner et al. 1996). Unlike patientswith arthrogryposis, however, clp/clp mice display nonfixed ab-normal limb postures in early life. Very severely affected mutantsdie within 2–3 days after birth, but those that survive the firstseveral weeks have a normal life span. The claw paw gene, whichis molecularly undefined, has been mapped by linkage analysisclose to the glucose phosphate isomerase-1 locus (Gpi-1)onmouse Chromosome (Chr) 7 (Henry et al. 1991).Myelin-associated glycoprotein (MAG) is a myelin-specificcell adhesion molecule of the immunoglobulin super-family ofmembrane proteins (Arquint et al. 1987; Lai et al. 1987). Themurine Mag gene has been mapped to mouse Chr (Barton et al.1987) where it is also closely linked to the Gpi-1locus. The MAGprotein is a minor constituent of CNS and PNS myelin, and itsdistribution at the glial-axonal interface suggests a specific role inthe early steps of axonal recognition and myelin formation.In myelinating glia, two MAG isoforms (termed S-MAG andL-MAG) are generated by alternative RNA splicing of exon 12,which carries an in-frame termination codon (Lai et al. 1987). Theresulting MAG-mRNAs encode a 67-kDa (S-MAG) and a 72-kDaisoform (L-MAG), which differ by the length of their carboxyltermini (Milner et al. 1990). In the rodent brain, these two MAGisoforms are developmentally regulated, with L-MAG being themajor neonatal form. In the peripheral nervous system, S-MAG isthe major form at all developmental stages.In an attempt to demonstrate the function of MAG in vivo, anull allele of the Mag gene has been created by gene targeting(Montag et al. 1994; Li et al. 1994). Surprisingly, MAG-deficientmouse mutants displayed no obvious developmental defects, ex-cept for subtle ultrastructural abnormalities of myelin, in markedcontrast to the dysmyelinated phenotype of claw paw, which isthus highly unlikely to be a functional Mag null allele. It remainsunknown which myelin proteins are able to compensate for theabsence of MAG in this null allele. In contrast, transgenic over-expression of the murine Mag gene may not be tolerated (M.Klugmann, A. Schneider, D. Montag, and K.-A. Nave, unpub-lished observation). However, even in mice with 8–10 transgeniccopies of the wild-type MAG gene, neurological deficits are rela-tively subtle and the clinical phenotype is transient and differentfrom that of claw paw mice. Nevertheless, MAG overexpression inmice suggested to us that alterations of this protein may result inaberrant gain-of-function effects. A well-documented example thatmissense mutations can underlie a severe dysmyelination (whereasthe complete deletion of the same gene is compatible with myelinassembly) is provided by the proteolipid protein (PLP) gene, en-coding the major component of CNS myelin (Klugmann et al.1997). Given the close linkage between Mag and clp (see below),Mag qualified as a candidate gene for the claw paw mutation:alterations of the MAG coding sequence could, for example, exerta misfolded protein with dominant-negative effects on other mem-brane proteins, yet without having the full penetrance of an auto-somal-dominant mutation. In fact, heterozygous claw paw miceare viable, but some myelin abnormalities have been described(Henry et al. 1991).A search of all linkage data available from the Mouse GenomeDatabase (Jackson Laboratory), comprising a total of 332 mice,did not show any recombination between Mag and Gpi-1(Rudert1992; Saunders 1990; Tong 1993; Fig. 1A). Since clp is alsoclosely linked to Gpi-1(Henry et al. 1991), the physical proximityof Mag and clp can be inferred. To test directly the possible in-volvement of MAG in the claw paw defect, we determined thecDNA-sequence of both MAG-isoforms in mice homozygous forthe clp mutation. Assuming that peripheral and central MAG pro-teins are molecularly indistinguishable, cDNA was reverse-transcribed from total brain RNA of a visibly affected mutant andan age-matched wild-type control. In independent amplifications,which followed standard procedures, three overlapping fragmentsof the MAG cDNA were generated, including 47 bp at the 58 endand 14 bp at the 38 end of the open reading frame (Fujita et al.1989), with different primer pairs corresponding to the knownexon sequences (Fig 1B). The PCR-generated products had theexpected sizes of 433 bp (with primer pair E3-AS6), 394 bp(primer pair S6-AS8), and 980 bp (primer pairS8-E13). RT-PCR

P19 embryonal carcinoma cells differentiate into neuroectodermal derivates upon aggregation and treatment with retinoic acid (RA). Such RA-induced P19 cells, concomitant with neuronal differentiation, produce the neuropeptide head activator (HA) and release HA into the culture medium. The amount of HA produced by such differentiating P19 cells is more than 100-fold higher than that of uninduced P19 cells. RA-induced P19 cells respond to HA treatment with a two-fold increase in total number of neuronally differentiating P19 progeny. As early response to HA, an increase in intracellular calcium concentration is observed.