Phenotypic analysis of mice deficient in the major myelin protein MOBP, and evidence for a novel Mobp isoform.

Myelin-associated oligodendrocytic basic protein (MOBP) is a recently identified major component of central nervous system (CNS) myelin. We previously reported a detailed characterization of the genomic region encompassing the Mobp gene, elucidating the complex series of transcript splicing responsible for the generation of its diverse family of protein isoforms. These basic, positively charged polypeptides display spatial and temporal expression patterns consistent with a potential role in the compaction and maintenance of the myelin sheath. MOBP isoforms have also been localized to the nucleus and the microtubular network of oligodendrocytes; transcript corresponding to one isoform is present during embryonic development. Recent reports have identified a role for this protein family in the pathogenesis of multiple sclerosis, but a clear function for the wild-type protein has remained unclear. We report a detailed analysis of a targeted mutation of Mobp, which results in the deletion of the translational start site and most of the coding sequence of MOBP, and the deletion of the entire coding sequence corresponding to a novel, putative MOBP isoform. Our analyses clearly demonstrate that MOBP-deficient mice develop normally, generate intact compact CNS myelin, and demonstrate no obvious clinical phenotype. Furthermore, in contrast with another recent study, we find that Mobp null mice demonstrate no significant influence on the axonal diameter of myelinated axons. Although MOBP is not essential for myelination, it appears that its absence is not simply compensated for by increased expression of the "classic" myelin basic protein (MBP).

EDNRB/EDN3 and Hirschsprung disease type II.

The study of vertebrate pigmentary anomalies has greatly improved our understanding of melanocyte biology. One such disorder, Waardenburg syndrome (WS), is a mendelian trait characterized by hypopigmentation and sensorineural deafness. It is commonly subdivided into four types (WS1-4), defined by the presence or absence of additional symptoms. WS type 4 (WS4), or Shah-Waardenburg syndrome, is also known as Hirschsprung disease Type II (HSCR II) and is characterized by an absence of epidermal melanocytes and enteric ganglia. Mutations in the genes encoding the endothelin type-B receptor (EDNRB) and its physiological ligand endothelin 3 (EDN3) are now known to account for the majority of HSCR II patients. Null mutations in the mouse genes Ednrb and Edn3 have identified a key role for this pathway in the normal development of melanocytes and other neural crest-derived lineages. The pleiotropic effects of genes in this pathway, on melanocyte and enteric neuron development, have been clarified by the embryologic identification of their common neural crest (NC) ancestry. EDNRB and EDN3 are transiently expressed in crest-derived melanoblast and neuroblast precursors, and in the surrounding mesenchymal cells, respectively. The influence of EDNRB-mediated signaling on the emigration, migration, proliferation, and differentiation of melanocyte and enteric neuron precursors, in vivo and in vitro has recently been the subject of great scrutiny. A major emergent theme is that EDN3-induced signaling prevents the premature differentiation of melanocyte and enteric nervous system precursors and is essential between 10 and 12.5 days post-coitum. We review the present understanding of pigment cell development in the context of EDNRB/EDN3--a receptor-mediated pathway with pleiotropic effects.

Splicing pattern, transcript start distribution, and DNA sequence of the mouse gene (Mobp) encoding myelin-associated oligodendrocytic basic protein.

We have cloned the mouse gene Mobp, encoding the family of myelin-associated oligodendrocytic basic proteins (MOBP), to facilitate elucidation of its genomic organization and regulation. We report near complete sequence analysis of the Mobp gene (>11 kb), including complete sequence of all exons and their associated splice junctions. The Mobp gene comprises eight discrete exons and encompasses a genomic region in excess of 15 kb. We provide a definitive analysis of the alternative splicing events and exon usage required in the generation of the reported splice variants of Mobp transcripts. We identify sequences corresponding to the coding regions of all reported protein isoforms. Consequently, we demonstrate that sequence regions, predicted to encode unique portions of two putative protein isoforms in the rat (MOBP 71 and MOBP 99), are not fully conserved between the rat and the mouse: we predict that the mouse equivalents are two distinct polypeptides of 73 amino acids, MOBP73A and MOBP73B, respectively. We have analyzed sequence from 63 oligo-capped, cloned cDNA fragments and identify six transcription start points associated with the Mobp gene at postnatal day 26. This study provides the platform for a more detailed analysis of the function of the Mobp gene product and subsequent evaluation of its possible involvement in known neuropathies.

Reduced levels of a specific myelin-associated oligodendrocytic basic protein isoform in shiverer myelin.

Myelin-associated oligodendrocytic basic protein (MOBP) and myelin basic protein (MBP) share many structural similarities. MOBP is synthesised by mature oligodendrocytes and localised at the major dense line (MDL), suggesting a role in the myelin compaction process. The shiverer mouse, a deletion mutant of the myelin basic protein (Mbp) gene, has poorly compacted myelin with essentially no MDL. In this study we compare the developmental expression of the Mobp gene in wild-type and shiverer mice. The significant finding is that one of the two abundant MOBP isoforms, the approximately 20-kD species, is poorly incorporated into shiverer myelin. The absence is specific to shiverer and is not a feature of dysmyelinating mutants with an abnormal intraperiod line. Our data suggest that incorporation of this MOBP isoform into shiverer myelin may be influenced by the presence of MBP or be a consequence of a disrupted MDL.

Cytoskeletal and nuclear localization of myelin oligodendrocytic basic protein isoforms.

The recently described single copy myelin-associated oligodendrocytic basic protein (Mobp) gene is expressed exclusively in the central nervous system (CNS). The gene encodes a family of small highly basic polypeptides with predicted amino acid lengths of 69, 71, 81, 99 and 170, all of which share a 68 residue amino terminal. Here we report on the subcellular distribution of two of these polypeptides termed MOBP81 and MOBP170 in transiently transfected Cos7 cells using an antibody raised against a region common to all isoforms of MOBP. Additionally, we describe MOBP trafficking in cultured mouse spinal cord oligodendrocytes. Immunostaining for MOBP81 is intense in the perinuclear region and extends throughout the cytoplasm colocalizing with the microtubular cytoskeletal network. Consistent with this we demonstrate that MOBP partitions with the cytoskeletal fraction prepared from myelin. In contrast, although MOBP170 is present in the cytoplasm it does not colocalize with the cytoskeleton and displays a greater variation in distribution. In the majority of transfectants immunostaining is present throughout the karyoplasm but with increased intensity around the nucleolus. Within mouse primary oligodendrocytes endogenous MOBP is present in the cell body and processes colocalizing with the microtubular network. Immunoreactivity is not detectable in the nucleus in these mature oligodendrocytes. These significant differences in MOBP81 and MOBP170 protein kinesis coupled to different expression profiles of their respective message populations may be indicative of both myelin structural and cellular/regulatory functions, respectively, for these polypeptides.

Developmental expression of the murine Mobp gene.

In this report we describe the developmental expression of the murine (Mobp) gene encoding myelin-associated oligodendrocytic basic protein. We have characterized three Mobp cDNA clones which have been used as probes. Murine Mobp splice variant-1 (mmsv-1), a portion of 3' untranslated region (UTR), is homologous to 3' UTR sequences found in the rat Mobp splice variants rOP1, Mobp81-A and Mobp-99. The mmsv-2 sequence, encoding 81 amino acids, closely resembles the rat Mobp81-A splice variant. The mmsv-3 cDNA, encoding 170 amino acids corresponding closely to the rat rOPRP1 splice variant, detects a single mRNA species present in low levels from E12 onward, suggesting this MOBP may have a function alternative or additional to involvement in myelin formation. The mmsv-1 probe detects an mRNA species abundantly expressed in the postnatal central nervous system (CNS) but barely detectable at E18. This mRNA is located initially in the cell bodies of oligodendrocytes, moving distally into their processes as myelination proceeds. The most abundant mmsv(s) in the adult CNS are present at detectable levels after expression of the myelin basic protein (Mbp) gene and marginally after or coincident with the proteolipid protein (Plp) gene. The level of the abundant, late-expressed mRNA correlates closely with the capacity to form myelin and the maturity of oligodendrocytes, as shown in two hypomyelinated mutants, rumpshaker and jimpy, which represent mildly and severely affected phenotypes, respectively.