Peripheral nerve myelin facilitates rapid impulse conduction and normal motor and sensory functions. Many aspects of myelin biogenesis, glia-axonal interactions, and nerve homeostasis are poorly understood at the molecular level. We therefore hypothesized that only a fraction of all relevant myelin proteins has been identified so far. Combining gel-based and gel-free proteomic approaches, we identified 545 proteins in purified mouse sciatic nerve myelin, including 36 previously known myelin constituents. By mass spectrometric quantification, the predominant P0, periaxin, and myelin basic protein constitute 21, 16, and 8% of the total myelin protein, respectively, suggesting that their relative abundance was previously misestimated due to technical limitations regarding protein separation and visualization. Focusing on tetraspan-transmembrane proteins, we validated novel myelin constituents using immunobased methods. Bioinformatic comparison with mRNA-abundance profiles allowed the categorization in functional groups coregulated during myelin biogenesis and maturation. By differential myelin proteome analysis, we found that the abundance of septin 9, the protein affected in hereditary neuralgic amyotrophy, is strongly increased in a novel mouse model of demyelinating neuropathy caused by the loss of prion protein. Finally, the systematic comparison of our compendium with the positions of human disease loci allowed us to identify several candidate genes for hereditary demyelinating neuropathies. These results illustrate how the integration of unbiased proteome, transcriptome, and genome data can contribute to a molecular dissection of the biogenesis, cell biology, metabolism, and pathology of myelin. Peripheral nerve myelin facilitates rapid impulse conduction and normal motor and sensory functions. Many aspects of myelin biogenesis, glia-axonal interactions, and nerve homeostasis are poorly understood at the molecular level. We therefore hypothesized that only a fraction of all relevant myelin proteins has been identified so far. Combining gel-based and gel-free proteomic approaches, we identified 545 proteins in purified mouse sciatic nerve myelin, including 36 previously known myelin constituents. By mass spectrometric quantification, the predominant P0, periaxin, and myelin basic protein constitute 21, 16, and 8% of the total myelin protein, respectively, suggesting that their relative abundance was previously misestimated due to technical limitations regarding protein separation and visualization. Focusing on tetraspan-transmembrane proteins, we validated novel myelin constituents using immuno-based methods. Bioinformatic comparison with mRNA-abundance profiles allowed the categorization in functional groups coregulated during myelin biogenesis and maturation. By differential myelin proteome analysis, we found that the abundance of septin 9, the protein affected in hereditary neuralgic amyotrophy, is strongly increased in a novel mouse model of demyelinating neuropathy caused by the loss of prion pr...

In the peripheral nervous system, the 'trigger' of myelination is axon size (>1µm), a threshold mediated by Nrg1 type III on the axon surface 1 . Since Nrg1 is not required for CNS myelination 2 , we asked whether axon size can be experimentally increased here by the absence of PTEN 3 , i.e. with PI(3,4,5)P3 stimulating the AKT1/mTOR pathway ( Fig.1a). To specifically enlarge cerebellar granule cells (GC) and their naturally unmyelinated parallel fiber (Pf) axons, we generated Tg (m6) (Fig.1d).Mutant mice appeared healthy (Supplementary Video1), but cerebellar GC layer (GL) and molecular layer (ML) progressively enlarged ( Fig.1e; Supplementary Fig.1a).Immunostaining of GABAA receptor 6 revealed a gradual size increase of GC somata ( Supplementary Fig.1b). Older mice developed ataxia and tremor (Supplementary Video2), possibly due to hamartomas at advanced age (Supplementary Fig.2; Supplementary Information).The diameter of Pf also increased over time, as quantified by electron microscopy (EM), reaching 0.61±0.009µm in mutants over 0.16±0.002 µm in controls at 1 year of age ( Fig.1f,g; Supplementary Fig.1c). Importantly, radial Pf growth in mutant brains was associated with significant myelination beginning at age P40 (2.30.6% myelinated Pf) and progressing over time (3 months: 9.51% myelinated Pf), up to 3 403% of Pf being myelinated at 1 year with an average g-ratio of 0.84. Myelination was visualized by Gallyas silver impregnation ( Supplementary Fig.3a), anti-CNP immunohistochemistry ( Fig.1h, Supplementary Fig.3b), including whole-mount immunolabelling combined with light sheet and 2-photon microscopy (Supplementary Video3), as well as EM (Fig.1i-k). We determined a 'size threshold' of approximately 0.25 µm for myelination at all ages tested (Fig.1k,l). Myelinated Pf segments were very rarely seen in controls.De novo myelination included the formation of nodes and paranodes with septate -like junctions ( Fig.1j) and the clustering of Caspr and NaV1.6 on axons (Fig.1m,n). GC synaptic contacts to Purkinje cell dendrites were restricted to Pf 'nodal' regions ( Fig.1o; Supplementary Fig.4).The ML harbors normally very few oligodendrocytes and only scattered OPC s, as demonstrated in Plp1-DsRed*Ng2-EYFP double-transgenic mice (Fig.2a). In Pten mutants at P45, mature oligodendrocytes were increased in the GL (+33%) and more prominent in the ML (+750%), yet unaltered in cerebellar white matter (WM) (Fig.2b).To determine whether OPCs proliferate in the ML or are recruited from the GL, we combined immunostaining with BrdU labeling (Fig.2c). After 20 days of daily BrdU injections (P25-45), OPC proliferation (BrdU+,Olig2+) was the same in GL or WM of mutants and controls (Fig.2d). However, OPC proliferation in the mutant ML was 4.4 -fold increased. Even at age 6-7 months, we found a 4.7-fold increase of BrdU+,Olig2+ cells. This suggests that local proliferation is stimulated (directly or indirectly) by GC axon-derived signals, which must still be present at older ages. At that time, oligodendrocyte lineage cel...

Establishment of long-range fiber tracts by neocortical projection neurons is fundamental for higher brain functions. The molecular control of axon tract formation, however, is still poorly understood. Here, we have identified basic helix-loop-helix (bHLH) transcription factors Neurod2 and Neurod6 as key regulators of fasciculation and targeted axogenesis in the mouse neocortex. In Neurod2/6 double-mutant mice, callosal axons lack expression of the cell adhesion molecule Contactin2, defasciculate in the subventricular zone, and fail to grow toward the midline without forming Probst bundles. Instead, mutant axons overexpress Robo1 and follow random trajectories into the ipsilateral cortex. In contrast to long-range axogenesis, generation and maintenance of pyramidal neurons and initial axon outgrowth are grossly normal, suggesting that these processes are under distinct transcriptional control. Our findings define a new stage in corpus callosum development and demonstrate that neocortical projection neurons require transcriptional specification by neuronal bHLH proteins to execute an intrinsic program of remote connectivity.

Short interspersed repetitive elements (SINEs) are highly repeated sequences that account for a significant proportion of many eukaryotic genomes and are usually considered “junk DNA”. However, we previously discovered that many AmnSINE1 loci are evolutionarily conserved across mammalian genomes, suggesting that they may have acquired significant functions involved in controlling mammalian-specific traits. Notably, we identified the AS021 SINE locus, located 390 kbp upstream of Satb2. Using transgenic mice, we showed that this SINE displays specific enhancer activity in the developing cerebral cortex. The transcription factor Satb2 is expressed by cortical neurons extending axons through the corpus callosum and is a determinant of callosal versus subcortical projection. Mouse mutants reveal a crucial function for Sabt2 in corpus callosum formation. In this study, we compared the enhancer activity of the AS021 locus with Satb2 expression during telencephalic development in the mouse. First, we showed that the AS021 enhancer is specifically activated in early-born Satb2+ neurons. Second, we demonstrated that the activity of the AS021 enhancer recapitulates the expression of Satb2 at later embryonic and postnatal stages in deep-layer but not superficial-layer neurons, suggesting the possibility that the expression of Satb2 in these two subpopulations of cortical neurons is under genetically distinct transcriptional control. Third, we showed that the AS021 enhancer is activated in neurons projecting through the corpus callosum, as described for Satb2+ neurons. Notably, AS021 drives specific expression in axons crossing through the ventral (TAG1−/NPY+) portion of the corpus callosum, confirming that it is active in a subpopulation of callosal neurons. These data suggest that exaptation of the AS021 SINE locus might be involved in enhancement of Satb2 expression, leading to the establishment of interhemispheric communication via the corpus callosum, a eutherian-specific brain structure.

Acquisition of cis-regulatory elements is a major driving force of evolution, and there are several examples of developmental enhancers derived from transposable elements (TEs). However, it remains unclear whether one enhancer element could have been produced via cooperation among multiple, yet distinct, TEs during evolution. Here we show that an evolutionarily conserved genomic region named AS3_9 comprises three TEs (AmnSINE1, X6b_DNA and MER117), inserted side-by-side, and functions as a distal enhancer for wnt5a expression during morphogenesis of the mammalian secondary palate. Functional analysis of each TE revealed step-by-step retroposition/transposition and co-option together with acquisition of a binding site for Msx1 for its full enhancer function during mammalian evolution. The present study provides a new perspective suggesting that a huge variety of TEs, in combination, could have accelerated the diversity of cis-regulatory elements involved in morphological evolution.