OBJECTIVE:Repair of myelin injury in multiple sclerosis may fail, resulting in chronic demyelination, axonal loss, and disease progression. As cellular pathways regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN; eg, phosphatidylinositol-3-kinase [PI-3K]) have been reported to enhance axon regeneration and oligodendrocyte maturation, we investigated potentially beneficial effects of Pten loss of function in the oligodendrocyte lineage on remyelination. METHODS:We characterized oligodendrocyte numbers and myelin sheath thickness in mice with conditional inactivation of Pten in oligodendrocytes, Olig2-cre, Pten(fl/fl) mice. Using a model of central nervous system demyelination, lysolecithin injection into the spinal cord white matter, we performed short- and long-term lesioning experiments and quantified oligodendrocyte maturation and myelin sheath thickness in remyelinating lesions. RESULTS:During development, we observed dramatic hypermyelination in the corpus callosum and spinal cord. Following white matter injury, however, there was no detectable improvement in remyelination. Moreover, we observed progressive myelin sheath abnormalities and massive axon degeneration in the fasciculus gracilis of mutant animals, as indicated by ultrastructure and expression of SMI-32, amyloid precursor protein, and caspase 6. INTERPRETATION:These studies indicate adverse effects of chronic Pten inactivation (and by extension, activation PI-3K signaling) on myelinating oligodendrocytes and their axonal targets. We conclude that PTEN function in oligodendrocytes is required to regulate myelin thickness and preserve axon integrity. In contrast, PTEN is dispensable during myelin repair, and its inactivation confers no detectable benefit.

The progressive loss of CNS myelin in patients with multiple sclerosis (MS) has been proposed to result from the combined effects of damage to oligodendrocytes and failure of remyelination. A common feature of demyelinated lesions is the presence of oligodendrocyte precursors (OLPs) blocked at a premyelinating stage. However, the mechanistic basis for inhibition of myelin repair is incompletely understood. To identify novel regulators of OLP differentiation, potentially dysregulated during repair, we performed a genome-wide screen of 1040 transcription factor-encoding genes expressed in remyelinating rodent lesions. We report that approximately 50 transcription factor-encoding genes show dynamic expression during repair and that expression of the Wnt pathway mediator Tcf4 (aka Tcf7l2) within OLPs is specific to lesioned-but not normal-adult white matter. We report that beta-catenin signaling is active during oligodendrocyte development and remyelination in vivo. Moreover, we observed similar regulation of Tcf4 in the developing human CNS and lesions of MS. Data mining revealed elevated levels of Wnt pathway mRNA transcripts and proteins within MS lesions, indicating activation of the pathway in this pathological context. We show that dysregulation of Wnt-beta-catenin signaling in OLPs results in profound delay of both developmental myelination and remyelination, based on (1) conditional activation of beta-catenin in the oligodendrocyte lineage in vivo and (2) findings from APC(Min) mice, which lack one functional copy of the endogenous Wnt pathway inhibitor APC. Together, our findings indicate that dysregulated Wnt-beta-catenin signaling inhibits myelination/remyelination in the mammalian CNS. Evidence of Wnt pathway activity in human MS lesions suggests that its dysregulation might contribute to inefficient myelin repair in human neurological disorders.

Within the motoneuron precursor (pMN) domain of the developing spinal cord, the bHLH transcription factor, Olig2, plays critical roles in pattern formation and the generation of motor neuron and oligodendrocyte precursors. How are the multiple functions of Olig2 regulated? We have isolated a large BAC clone encompassing the human OLIG2 locus that rescues motor neuron and oligodendrocyte development but not normal pattern formation in Olig2−/− embryos. Within the BAC clone, we identified a conserved 3.6 kb enhancer sub-region that directs reporter expression specifically in the motor neuron lineage but not oligodendrocyte lineage in vivo. Our findings indicate complex regulation of Olig2 by stage- and lineage-specific regulatory elements. They further suggest that transcriptional regulation of Olig2 is involved in segregation of pMN neuroblasts.

We investigated whether co-expression of Neurog 1 and Atoh 1 in common neurosensory precursors could explain the loss of hair cells in Neurog 1 null mice. Analysis of terminal mitosis, using BrdU, supports previous findings regarding timing of exit from cell cycle. Specifically, we show that cell cycle exit occurs in spiral sensory neurons in a base-to-apex progression followed by cell cycle exit of hair cells in the organ of Corti in an apex-to-base progression, with some overlap of cell cycle exit in the apex for both hair cells and spiral sensory neurons. Hair cells in Neurog 1 null mice show cell cycle exit in an apex-to-base progression about 1-2 days earlier. Atoh 1 is expressed in an apex-to-base progression rather then a base-to-apex progression as in wildtype littermates. We tested the possible expression of Atoh1 in neurosensory precursors using two Atoh 1-Cre lines. We show Atoh 1-Cre mediated beta-galactosidase expression in delaminating sensory neuron precursors as well as undifferentiated epithelial cells at E11 and E12.5. PCR analysis shows expression of Atoh 1 in the otocyst as early as E10.5, prior to any histology-based detection techniques. Combined, these data suggest that low levels of Atoh 1 exist much earlier in precursors of hair cells and sensory neurons, possibly including neurosensory precursors. Analysis of Atoh 1-Cre expression in E18.5 embryos and P31 mice reveal beta-galactosidase stain in all hair cells but also in vestibular and cochlear sensory neurons and some supporting cells. A similar expression of Atoh 1-LacZ exists in postnatal and adult vestibular and cochlear sensory neurons, and Atoh 1 expression in vestibular sensory neurons is confirmed with RT-PCR. We propose that the absence of NEUROG 1 protein leads to loss of sensory neuron formation through a phenotypic switch of cycling neurosensory precursors from sensory neuron to hair cell fate. Neurog 1 null mice show a truncation of clonal expansion of hair cell precursors through temporally altered terminal mitosis, thereby resulting in smaller sensory epithelia.

Olig1 and Olig2 are closely related basic helix-loop-helix (bHLH) transcription factors that are expressed in myelinating oligodendrocytes and their progenitor cells in the developing central nervous system (CNS). Olig2 is necessary for the specification of oligodendrocytes, but the biological functions of Olig1 during oligodendrocyte lineage development are poorly understood. We show here that Olig1 function in mice is required not to develop the brain but to repair it. Specifically, we demonstrate a genetic requirement for Olig1 in repairing the types of lesions that occur in patients with multiple sclerosis.

During development of the elegantly structured mammalian cerebral cortex, generation and subsequent migration of specialized neurons and glia within the brain follows a characteristic sequence. The earliest differentiated neurons emigrate from the telencephalic ventricular zone to form a superficial primordial layer called the preplate (Allendoerfer and Shatz, 1994). In mice, the marginal zone (MZ, layer I) and subplate (SP) derive from splitting of the preplate at ~11-12.5 days postcoitum (dpc) as successive waves of neurons from the underlying ventricular zone undergo radial migration into the cortical plate proper (layers II-VI). The MZ and SP comprise a heterogeneous population of cells with neuronal properties, including reelin-expressing Cajal-Retzius (CR) cells, subpial granule cells (glutamate-positive) and subpallial-derived interneurons (GABA-positive) (Fairen et al., 2002; Meyer et al., 1998; Parnavelas, 2000; Zecevic et al., 1999). Important roles for MZ and SP cells during tangential and radial migration of cortical neurons have been previously described (Parnavelas, 2000; Xie et al., 2002). For example, proper formation of the MZ and SP has been shown to be necessary for tangential emigration of GABA-expressing interneurons from ventral forebrain regions (e.g. the ganglionic eminence) into the cortical plate (Lavdas et al., 1999). Second, cells in the MZ provide repulsive or stop cues to radially migrating cortical 2275 Development 130, 2275-2287 © 2003 The Company of Biologists Ltd doi:10.1242/dev.00421

Leptomeningeal glioneuronal heterotopias are a focal type of cortical dysplasia in which neural cells migrate aberrantly into superficial layers of the cerebral cortex and meninges. These heterotopias are frequently observed as microscopic abnormalities in the brains of individuals with central nervous system (CNS) malformations and epilepsy. Previous work has demonstrated that the function of Emx2, which encodes a homeodomain transcription factor, is essential for development of the cortical preplate, which gives rise to the marginal zone and subplate. However, transcriptional targets of EMX2 during CNS development are unknown. We report that leptomeningeal glioneuronal heterotopias form in Emx2–/– mice that are equivalent to human lesions. Additionally, we observed ectopic expression of Wnt1 in the embryonic roofplate organizer region and dorsal telencephalon. To determine the phenotypic consequences of such Wnt1 misexpression, we deleted a putative EMX2 DNA-binding site from the Wnt1 enhancer and used this to misexpress Wnt1 in the developing murine CNS. Heterotopias were detected in transgenic mice as early as 13.5 days postcoitum, consistent with a defect of preplate development during early phases of radial neuronal migration. Furthermore, we observed diffuse abnormalities of reelin- and calretinin-positive cell populations in the marginal zone and subplate similar to those observed in Emx2-null animals. Taken together, these findings indicate that EMX2 is a direct repressor of Wnt1 expression in the developing mammalian telencephalon. They further suggest that EMX2-Wnt1 interactions are essential for normal development of preplate derivatives in the mammalian cerebral cortex.

BACKGROUND:Organizing signals such as Sonic hedgehog are thought to specify neuronal subtype identity by regulating the expression of homeodomain proteins in progenitors of the embryonic neural tube. One of these, Nkx2.2, is necessary and sufficient for the development of V3 interneurons.RESULTS:We report that Olig genes, encoding basic helix-loop-helix (bHLH) proteins, are expressed in a subset of Nkx2.2 progenitors before the establishment of interneurons and oligodendroglial precursors. Gain-of-function analysis in transgenic mouse embryos indicates that Olig genes specifically inhibit the establishment of Sim1-expressing V3 interneurons. Moreover, coexpression of Olig2 with Nkx2.2 in the chick neural tube generated cells expressing Sox10, a marker of oligodendroglial precursors. Colocalization of Olig and Nkx2.2 proteins at the dorsal extent of the Nkx2.2 expression domain is consistent with regulatory interactions that define the potential of progenitor cells in the border region.CONCLUSIONS:Interactions between homeodomain and Olig bHLH proteins evidently regulate neural cell fate acquisition and diversification in the ventral neural tube. In particular, interactions between Olig and Nkx2.2 proteins inhibit V3 interneuron development and promote the formation of alternate cell types, including those expressing Sox10.