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Granule neuron precursors (GNPs) are the most actively proliferating ceils in the postnatal nervous system, and mutations in pathways that control the GNP ceil cycle can result in medulloblastoma. The transcription factor Atoh1 has been suspected to contribute to GNP proliferation, but its role in normal and neoplastic postnatal cerebellar development remains unexplored. We show that Atoh1 regulates the signal transduction pathway of Sonic Hedgehog, an extracellular factor that is essential for GNP proliferation, and demonstrate that deletion of Atoh1 prevents cerebellar neoplasia in a mouse model of medulloblastoma. Our data shed Light on the function of Atoh1 in postnatal cerebellar development and identify a new mechanism that can be targeted to regulate medulloblastoma formation. doi: 10.1126/science.1181453

There are more than 50 class I MHC (MHCI) molecules in the mouse genome, some of which are now known to be expressed in neurons; however, the role of classical MHCI molecules in synaptic plasticity is unknown. We report that the classical MHCI molecules, H2-[K.sup.b] and H2-[D.sup.b], are co-expressed by Purkinje cells (PCs). In the cerebellum of mice deficient for both H2-[K.sup.b] and H2-[D.sup.b] ([K.sub.b][D.sub.b-/-]), there is a lower threshold for induction of long-term depression (LTD) at parallel fiber to PC synapses. This change may be a result of additional glutamate release observed at [K.sub.b][D.sub.b-/-] CF to PC synapses, which are thought to 'train' the cerebellar circuit. A behavioral correlate of cerebellar LTD is motor learning; acquisition and retention of a Rotarod behavioral task is significantly better in [K.sub.b][D.sub.b-/-] mice than in WT cohorts. These physiological and behavioral phenotypes in [K.sub.b][D.sub.b-/-] mice reveal a surprising role for classical MHCI molecules in synaptic plasticity and motor learning. cerebellum | neuronal MHC class 1 | synaptic plasticity

Inactivation of homologous recombination (HR) or nonhomologous end-joining (NHEJ) predisposes to a spectrum of tumor types. Here, we inactivated DNA double-strand break repair (DSBR) proteins, DNA Ligase IV (Lig4), Xrcc2, and Brca2, or combined Lig4/ Xrcc2 during neural development using Nestin-cre. In all cases, inactivation of these repair factors, together with p53 loss, led to rapid medulloblastoma formation. Genomic analysis of these tumors showed recurring chromosome 13 alterations via chromosomal loss or translocations involving regions containing Ptch1. Sequence analysis of the remaining Ptch1 allele showed a variety of inactivating mutations in all tumors analyzed, highlighting the critical tumor suppressor function of this hedgehog-signaling regulator. We also observed genomic amplification or up-regulation of either N-Myc or cyclin D2 in all medulloblastomas. Additionally, chromosome 19, which contains Pten, was also selectively deleted in medulloblastoma arising after disruption of HR. Thus, our data highlight the preeminence of Ptch1 as a tumor suppressor in cerebellar granule cells and reveal other genomic events central to the genesis of medulloblastoma. DNA strand breaks | patched1 | sonic hedgehog | cerebellum

The central dogma of radiation biology, that biological effects of ionizing radiation are a direct consequence of DNA damage occurring in irradiated cells, has been challenged by observations that genetic/ epigenetic changes occur in unexposed 'bystander cells' neighboring directly-hit cells, due to cell-to-cell communication or soluble factors released by irradiated cells. To date, the vast majority of these effects are described in cell-culture systems, while in vivo validation and assessment of biological consequences within an organism remain uncertain. Here, we describe the neonatal mouse cerebellum as an accurate in vivo model to detect, quantify, and mechanistically dissect radiation-bystander responses. DNA double-strand breaks and apoptotic cell death were induced in bystander cerebellum in vivo. Accompanying these genetic events, we report bystander-related tumor induction in cerebellum of radiosensitive Patched-1 (Ptch1) heterozygous mice after x-ray exposure of the remainder of the body. We further show that genetic damage is a critical component of in vivo oncogenic bystander responses, and provide evidence supporting the role of gap-junctional intercellular communication (GJIC) in transmission of bystander signals in the central nervous system (CNS). These results represent the first proof-of-principle that bystander effects are factual in vivo events with carcinogenic potential, and implicate the need for re-evaluation of approaches currently used to estimate radiation-associated health risks. cancer risk | DNA damage | in vivo| medulloblastoma | radiation

Neural stem cells and progenitors in the developing brain must choose between proliferation with renewal and differentiation. Defects in navigating this choice can result in malformations or cancers, but the genetic mechanisms that shape this choice are not fully understood. We show by positional cloning that the 30-zinc finger transcription factor Zfp423 (OAZ) is required for patterning the development of neuronal and glial precursors in the developing brain, particularly in midline structures. Mutation of Zfp423 results in loss of the corpus callosum, reduction of hippocampus, and a malformation of the cerebellum reminiscent of human Dandy-Walker patients. Within the cerebellum, Zfp423 is expressed in both ventricular and external germinal zones. Loss of Zfp423 results in diminished proliferation by granule cell precursors in the external germinal layer, especially near the midline, and abnormal differentiation and migration of ventricular zone-derived neurons and Bergmann gila. choroidplexus Ebf1 | nur12 Roaz | SMADs

Conditional N-Myc deletion limits the proliferation of granule neuron progenitors (GNPs), perturbs foliation, and leads to reduced cerebellar mass. We show that c-Myc mRNA levels increase in N-Myc-null GNPs and that simultaneous deletion of both c- and N-Myc exacerbates defective cerebellar development. Moreover, N-Myc loss has been shown to trigger the precocious expression of two cyclin-dependent kinase inhibitors, Kip1 and Ink4c, in the cerebellar primordium. We now further demonstrate that the engineered disruption of the Kip1 and Ink4c genes in N-Myc-null cerebella partially rescues GNP cell proliferation and cerebellar foliation. These results provide definitive genetic evidence that expression of N-Myc and concomitant down-regulation of Ink4c and Kip1 contribute to the proper development of the cerebellum. Myc | N-Myc deficiency | cerebellum | granule neuron progenitor | cell cycle inhibitor

Hedgehog pathway activation is required for proliferation of cerebellar granule cell neuron precursors during development and is etiologic in certain cerebellar tumors. To identify genes expressed specifically in granule cell neuron precursors, we used oligonucleotide microarrays to analyze regulation of 13,179 genes/expressed sequence tags in heterogeneous primary cultures of neonatal mouse cerebellum that respond to the mitogen Sonic hedgehog. In conjunction, we applied experiment-specific noise models to render a gene-by-gene robust indication of up-regulation in Sonic hedgehog-treated cultures. Twelve genes so identified were tested, and 10 (83%) showed appropriate expression in the external granular layer (EGL) of the postnatal day (PN) 7 cerebellum and down-regulation by PN 15, as verified by in situ hybridization. Whole-organ profiling of the developing cerebellum was carried out from PN 1 to 30 to generate a database of temporal gene regulation profiles (TRPs). From the database an algorithm was developed to capture the TRP typical of EGL-specific genes. The 'TRP-EGL' accurately predicted expression in vivo of an additional 18 genes/expressed sequence tags with a sensitivity of 80% and a specificity of 88%. We then compared the positive predictive value of our analytical procedure with other widely used methods, as verified by the TRP-EGL in silico. These findings suggest that replicate experiments and incorporation of noise models increase analytical specificity. They further show that genome-wide methods are an effective means to identify stage-specific gene expression in the developing granule cell lineage. Sonic hedgehog | proliferation | expression profiling | oligonucleotide microarray | noise

NeuroD, a bHLH transcription factor, is required for differentiation of the granule cells, postnatally generated microneurons, in the cerebellum and hippocampus. NeuroD-null mice, which die soon after birth from severe neonatal diabetes, were rescued by putting in a transgene that encodes the mouse neuroD (ital) gene under the insulin promoter. The mice still have severe neurological phenotype because of neuronal deficit in the granule layers of the cerebellum and hippocampus.

Elimination of excess climbing fiber (CF)Purkinje cell synapses during cerebellar development involves a signaling pathway that includes type 1 metabotropic glutamate receptor, G[Alpha]q, and the [Gamma] isoform of protein kinase C. To identify phospholipase C (PLC) isoforms involved in this process, we generated mice deficient in PLC[Beta]4, one of two major isoforms expressed in Purkinje cells. PLC[Beta]4 mutant mice are viable but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple CF innervation clearly is impaired in the rostral portion of the cerebellar vermis, in which PLC[Beta]4 mRNA is predominantly expressed. By contrast, CF synapse elimination is normal in the caudal cerebellum, in which low levels of PLC[Beta]4 mRNA but reciprocally high levels of PLC[Beta]3 mRNA are found. These results indicate that PLC[Beta]4 transduces signals that are required for CF synapse elimination in the rostral cerebellum.

The analysis of patterned gene expression has been an important tool for dissecting the molecular and developmental bases of functional compartmentalization in the mammalian cerebellum. In particular, sagittally-oriented cellular aggregates arranged along the mediolateral axis are the patterning element most commonly invoked to illustrate cerebellar compartmentalization, and these are revealed both by patterns of afferent projection and by a number of classical biochemical markers that are distributed in a pattern of 'zebra stripes'. Compartmentation along both the mediolateral and rostrocaudal axes might be linked mechanistically to segmentation in the fruit fly, since early cerebellar development is especially dependent upon the expression of mammalian homologs of Drosophila segmentation genes. In addition, as has been demonstrated in the retinotectal system, some of these genes are likely to control positional information required for the sagittal organization of cerebellar afferent projections. However, in contrast to these global or macro zones, the cerebellum is also compartmentalized at the subcellular or micro level. This can be visualized by differential patterns of mRNA distribution within the sole cerebellar efferent system, the Purkinje cell, defining within such cells a number of distinct subcellular domains or 'postal zones'. The global versus subcellular levels of cerebellar compartmentalization are related since they both appear to be linked to patterns of afferent innervation. A major goal of cerebellar research will be to unravel the true nature of such a relationship, and its relevance to function and behavior.

The development of two major subdivisions of the vertebrate nervous system, the midbrain and the cerebellum, is controlled by signals emanating from a constriction in the neural primordium called the midbrain/hindbrain organizer (Joyner, A. L. (1996) Trends Genet. 12, 15-201). The closely related transcription factors Pax-2 and Pax-5 exhibit an overlapping expression pattern very early in the developing midbrain/hindbrain junction. Experiments carried out in fish (Krauss, S., Maden, M., Holder, N. & Wilson, S. W. (1992) Nature (London) 360, 87-89) with neutralizing antibodies against Pax-b, the orthologue of Pax-2 in mouse, placed this gene family in an regulatory cascade necessary for the development of the midbrain and the cerebellum. The targeted mutation of Pax-5 has been reported to have only slight effects in the development of structures derived from the isthmic constriction, whereas the Pax-2 null mutant mice show a background-dependent phenotype with varying penetrance. To test a possible redundant function between Pax-2 and Pax-5 we analyzed the brain phenotypes of mice expressing different dosages of both genes. Our results demonstrate a conserved biological function of both proteins in midbrain/hindbrain regionalization. Additionally, we show that one allele of Pax-2, but not Pax.5, is necessary and sufficient for midbrain and cerebellum development in C57BL/6 mice.

Midbrain and cerebellum development depends on an organizing center that is located at the midbrain-hindbrain junction of the vertebrate embryo. Expression of the two closely related transcription factors Pax2 and Pax5 overlaps spatially and temporally in this region of the developing central nervous system. To study a possible interaction of these transcription factors in midbrain and cerebellum patterning, we have generated Pax5, Krd double mutant mice. The transgene-induced Krd mutation corresponds to an [approximately]7-centimorgan chromosome 19 deletion that eliminates the entire Pax2 locus. The heterozygous Krd mutation deleting one Pax2 allele had no effect on midbrain and cerebellum development. Moreover, only minor developmental defects were previously observed at the midline of the inferior colliculus and anterior cerebellum in mice that were homozygous for a targeted Pax5 mutation. Similar morphological alterations were observed in 80% of all compound heterozygous Pax5 (+/-) Krd (+/-) mice. However, in the remaining 20% of compound heterozygotes, the inferior colliculi were missing, and the vermis of the cerebellum was severely disrupted due to the failure of the cerebellar primordia to fuse at the midline. Inactivation of the second Pax5 allele in Pax5 (-/-) Krd (+/-) mice resulted in complete loss of the posterior midbrain and cerebellum, as the tissue originating from the midbrain-hindbrain boundary region was deleted in the embryo as early as day 9.5. On the basis of these data, we propose that the cooperation of Pax2 and Pax5 is essential for normal functioning of the organizing center at the midbrain-hindbrain junction.

We utilized a strain of mice, derived from a radiation mutagenesis experiment and carrying an activity-attenuated allele of the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD), to analyze the development of the cell lineage leading to cerebellar Purkinje neurons. Due to random X inactivation during early embryonic development, X-linked genes can be used to distinguish between clonally related populations of cells in X inactivation mosaics. Following histochemical staining for G6PD activity, the numeric proportions of Purkinje cells expressing either the wild-type or the mutant enzyme and the spatial distribution of these cellular phenotypes and their relation to anatomically and genetically defined cerebellar compartments were analyzed. Our data suggest that cerebellar Purkinje neurons originate from a limited pool of some 129 precursors. The size of this pool is different from the one derived from chimeric mice, allowing us to deduce the relative timing of Purkinje cell lineage restriction. Our data also show that Purkinje neurons of distinct lineage are extensively intermingled within the cerebellar cortex. Together, these findings suggest both a role for cell-cell communication in the development of genetically defined cerebellar compartments and a temporal window during which such cellular interactions may take place.