The pivotal role of irradiation-induced apoptosis in the pathogenesis and therapy of medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is a rare primitive neuroectodermal tumors originating from the cerebellum. MB is the most common malignant primary brain tumor of childhood. MB originates from neural precursor cells in distinctive regions of the rhombic lip, and their maturation occurs in the cerebellum or the brain stem during embryonal development. Also, apoptosis is a programmed cell death associated with numerous physiological as well as pathological regulations. RECENT FINDINGS: Irradiation (IR)-induce apoptosis triggers cell death, with or without intervening mitosis within a few hours of IR and these share different morphologic alteration such as, loss of normal nuclear structure as well as degradation of DNA. Moreover, MB is strikingly sensitive to DNA-damaging therapies and the role of apoptosis a key treatment modality. Furthermore, in MB, the apoptotic pathways are made up of several triggers, modulators, as well as effectors. Notably, IR-induced apoptotic mechanisms in MB therapy are very complex and they either induce radiosensitivity or inhibit radioresistance leading to potential effective treatment strategies for MB. CONCLUSION: This review explicitly explores the pivotal roles of IR-induced apoptosis in the pathogenesis and therapy of MB.

Cerebellar granule cell migration and folia development require Mllt11/Af1q/Tcf7c.

The organization of neurons into distinct layers, known as lamination, is a common feature of the nervous system. This process, which arises from the direct coupling of neurogenesis and neuronal migration, plays a crucial role in the development of the cerebellum, a structure exhibiting a distinct folding cytoarchitecture with cells arranged in discrete layers. Disruptions to neuronal migration can lead to various neurodevelopmental disorders, highlighting the significance of understanding the molecular regulation of lamination. We report a role Mllt11/Af1q/Tcf7c (myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 fused gene from chromosome 1q, also known as Mllt11 transcriptional cofactor 7; henceforth referred to Mllt11) in the migration of cerebellar granule cells (GCs). We now show that Mllt11 plays a role in both the tangential and radial migration of GCs. Loss of Mllt11 led to an accumulation of GC precursors in the rhombic lip region and a reduction in the number of GCs successfully populating developing folia. Consequently, this results in smaller folia and an overall reduction in cerebellar size. Furthermore, analysis of the anchoring centers reveals disruptions in the perinatal folia cytoarchitecture, including alterations in the Bergmann glia fiber orientation and reduced infolding of the Purkinje cell plate. Lastly, we demonstrate that Mllt11 interacts with non-muscle myosin IIB (NMIIB) and Mllt11 loss-reduced NMIIB expression. We propose that the dysregulation of NMIIB underlies altered GC migratory behavior. Taken together, the findings reported herein demonstrate a role for Mllt11 in regulating neuronal migration within the developing cerebellum, which is necessary for its proper neuroanatomical organization.

Human cerebellar organoids with functional Purkinje cells.

Research on human cerebellar development and disease has been hampered by the need for a human cell-based system that recapitulates the human cerebellum's cellular diversity and functional features. Here, we report a human organoid model (human cerebellar organoids [hCerOs]) capable of developing the complex cellular diversity of the fetal cerebellum, including a human-specific rhombic lip progenitor population that have never been generated in vitro prior to this study. 2-month-old hCerOs form distinct cytoarchitectural features, including laminar organized layering, and create functional connections between inhibitory and excitatory neurons that display coordinated network activity. Long-term culture of hCerOs allows healthy survival and maturation of Purkinje cells that display molecular and electrophysiological hallmarks of their in vivo counterparts, addressing a long-standing challenge in the field. This study therefore provides a physiologically relevant, all-human model system to elucidate the cell-type-specific mechanisms governing cerebellar development and disease.

Unified rhombic lip origins of group 3 and group 4 medulloblastoma.

Medulloblastoma, a malignant childhood cerebellar tumour, segregates molecularly into biologically distinct subgroups, suggesting that a personalized approach to therapy would be beneficial1. Mouse modelling and cross-species genomics have provided increasing evidence of discrete, subgroup-specific developmental origins2. However, the anatomical and cellular complexity of developing human tissues3-particularly within the rhombic lip germinal zone, which produces all glutamatergic neuronal lineages before internalization into the cerebellar nodulus-makes it difficult to validate previous inferences that were derived from studies in mice. Here we use multi-omics to resolve the origins of medulloblastoma subgroups in the developing human cerebellum. Molecular signatures encoded within a human rhombic-lip-derived lineage trajectory aligned with photoreceptor and unipolar brush cell expression profiles that are maintained in group 3 and group 4 medulloblastoma, suggesting a convergent basis. A systematic diagnostic-imaging review of a prospective institutional cohort localized the putative anatomical origins of group 3 and group 4 tumours to the nodulus. Our results connect the molecular and phenotypic features of clinically challenging medulloblastoma subgroups to their unified beginnings in the rhombic lip in the early stages of human development.

Failure of human rhombic lip differentiation underlies medulloblastoma formation.

Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain1-4. Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage5-8. By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL9,10. However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage3,4. Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2, CBFA2T3, PRDM6, UTX and OTX2. CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens, and G4 MB transcriptionally resembles these progenitors but are stalled in developmental time. Knockdown of OTX2 in model systems relieves this differentiation blockade, which allows MB cells to spontaneously proceed along normal developmental differentiation trajectories. The specific nature of the split human RL, which is destined to generate most of the neurons in the human brain, and its high level of susceptible EOMES+KI67+ unipolar brush cell progenitor cells probably predisposes our species to the development of G4 MB.

The interplay of atoh1 genes in the lower rhombic lip during hindbrain morphogenesis.

The Lower Rhombic Lip (LRL) is a transient neuroepithelial structure of the dorsal hindbrain, which expands from r2 to r7, and gives rise to deep nuclei of the brainstem, such as the vestibular and auditory nuclei and most posteriorly the precerebellar nuclei. Although there is information about the contribution of specific proneural-progenitor populations to specific deep nuclei, and the distinct rhombomeric contribution, little is known about how progenitor cells from the LRL behave during neurogenesis and how their transition into differentiation is regulated. In this work, we investigated the atoh1 gene regulatory network operating in the specification of LRL cells, and the kinetics of cell proliferation and behavior of atoh1a-derivatives by using complementary strategies in the zebrafish embryo. We unveiled that atoh1a is necessary and sufficient for specification of LRL cells by activating atoh1b, which worked as a differentiation gene to transition progenitor cells towards neuron differentiation in a Notch-dependent manner. This cell state transition involved the release of atoh1a-derivatives from the LRL: atoh1a progenitors contributed first to atoh1b cells, which are committed non-proliferative precursors, and to the lhx2b-neuronal lineage as demonstrated by cell fate studies and functional analyses. Using in vivo cell lineage approaches we revealed that the proliferative cell capacity, as well as the mode of division, relied on the position of the atoh1a progenitors within the dorsoventral axis. We showed that atoh1a may behave as the cell fate selector gene, whereas atoh1b functions as a neuronal differentiation gene, contributing to the lhx2b neuronal population. atoh1a-progenitor cell dynamics (cell proliferation, cell differentiation, and neuronal migration) relies on their position, demonstrating the challenges that progenitor cells face in computing positional information from a dynamic two-dimensional grid in order to generate the stereotyped neuronal structures in the embryonic hindbrain.

Quantitative study on human cerebellar cortex from anatomy cadaver preparations / Estudio cuantitativo de la corteza del cerebelo humano a partir de la anatomía de preparaciones cadavéricas

Human cerebellum is important for motor coordination; muscle tones and maintaing the equilibrium of the body. In our region, limited data is available on the normal morphology of human cerebellum, whilst fresh biopsy is quite difficult to obtain. Here adult male cerebellum from cadaver anatomy preparations embalmed with 37 % formalin fixative solution for over a year are studied (n=3). After removal, right cerebellum hemisphere was sliced into cubicle then temporary soaked into 50-60 % of alcohol before being paraffinated. Two parasagittal adjacent slices from each sample were deparaffinated (5 µm) and then stained with hematoxyllin-eosin (HE). Slides were observed under light microscope (Olympus, Japan). Pictures were analysed from 6 field numbers of each, with Optilab and Image Raster 3 software (Indonesia). The density of the Purkinje cells (Neuron purkinjense), the number and density of the Purkinje cells and the thickness of the molecular layer are measured. Data were analysed with the level of significance of p<0.05 (ANOVA, Microsoft Excel 2007). The distance between 2 Purkinje cells is ranged between 82.6-346.6 µm, although no significant differences found (p=0.1). There are no significant differences in the number and in the density of the Purkinje cells amongst samples (p=0.72 and 0.34, respectively); might be due to the similar age, sex and race of these cadavers. However, there is a significant difference in the thickness of the molecular layer (p=0.015). Variations amongst individual cerebelli are observed, with a significant different thickness in the molecular layer. The cellular composition of each cerebellum is unique, arguably correlated to the individual cerebellum activity when alive.

El cerebelo humano es importante en la coordinación motora, los tonos musculares y el mantenimiento del equilibrio del cuerpo. En nuestra región, son limitados los datos disponibles en relación a la morfología normal del cerebelo humano, por otra parte obtener una biopsia fresca es bastante difícil de obtener. Para este trabajo se utilizaron tres cerebelos adultos de sexo masculino, obtenidos a partir de cadáveres conservados con una solución de formalina al 37 % durante más de un año. Después de la separación, el hemisferio derecho del cerebelo fue seccionado en cubos que fueron posteriormente impregnados en una solución de 50-60 % de alcohol antes del proceso de parafinización. Se tomaron dos secciones parasagitales adyacentes (5 µm) de cada muestra desparafinada y luego éstas fueron teñidas con Hematoxilina-Eosina. Las muestras se observaron al microscopio óptico (Olympus, Japón). Se analizaron 6 campos por muestra con los softwares Optilab e Image Raster 3 (Indonesia). Se midieron la densidad de las neuronas purkinjenses (células de Purkinje), el número y densidad de ellas y el espesor de la capa molecular. Los datos fueron analizados con un nivel de significación de p<0,05 (ANOVA, Microsoft Excel 2007). La distancia entre dos células de Purkinje varió entre 82,6-346,6 µm, sin encontrarse diferencias significativas (p = 0,1). No hay diferencias significativas en el número (p = 0,72) y la densidad (p = 0,34) de células de Purkinje entre las muestras; podría deberse a la misma edad, el sexo y la raza de los cadáveres utilizados. Sin embargo, hay una diferencia significativa en el espesor de la capa molecular (p = 0,015). Se observaron variaciones individuales en el cerebelo, encontrándose diferencia significativa el espesor de la capa molecular. La composición celular del cerebelo es única, sin duda esta se correlaciona con la actividad del cerebelo en individuos vivos.

Ezh2 orchestrates topographic migration and connectivity of mouse precerebellar neurons.

We investigated the role of histone methyltransferase Ezh2 in tangential migration of mouse precerebellar pontine nuclei, the main relay between neocortex and cerebellum. By counteracting the sonic hedgehog pathway, Ezh2 represses Netrin1 in dorsal hindbrain, which allows normal pontine neuron migration. In Ezh2 mutants, ectopic Netrin1 derepression results in abnormal migration and supernumerary nuclei integrating in brain circuitry. Moreover, intrinsic topographic organization of pontine nuclei according to rostrocaudal progenitor origin is maintained throughout migration and correlates with patterned cortical input. Ezh2 maintains spatially restricted Hox expression, which, in turn, regulates differential expression of the repulsive receptor Unc5b in migrating neurons; together, they generate subsets with distinct responsiveness to environmental Netrin1. Thus, Ezh2-dependent epigenetic regulation of intrinsic and extrinsic transcriptional programs controls topographic neuronal guidance and connectivity in the cortico-ponto-cerebellar pathway.

Nuclear translocation of FGF8 and its implication to induce Sprouty2.

Fibroblast growth factor 8 (FGF8) functions as a local organizing signal for the tectum and cerebellum. FGF8 activates Ras-ERK signaling pathway to induce cerebellar development. We paid attention to the difference in the expression pattern of the molecules that are induced by FGF8 in the mid-hind brain region during normal development and after FGF8 misexpression; some are expressed in the area corresponding to the ERK activation domain but the others are expressed corresponding to the Fgf8 expression domain. Since some of the FGF family members are localized in the nucleus, we wondered if FGF8 could localize in the nuclei and function in the nucleus. We first show that in cultured NIH3T3 cells transfected FGF8b could localize in the nucleus. Transfected FGF8b could also localize in the nucleus of the cells in the chick neural tube. In mouse embryonic neural tube, we detected endogenous FGF8 in the nuclei. Implantation of an FGF8b-soaked bead showed that exogenous FGF8b could be translocated to the nuclei in the isthmus. Furthermore, signal-peptide-deletion mutant of FGF8b mainly localized in the nuclei, and induced Sprouty2 without activating ERK in the mesencephalon. Signal-peptide-deletion mutant of FGF8b could not induce Pax2 expression. Taken together, we concluded that FGF8b could be translocated to the nuclei, and that the nuclear FGF8 could function as transcriptional regulator to induce Sprouty2 in the isthmus.

Characterization of the neuropsychological phenotype of glycine N-methyltransferase-/- mice and evaluation of its responses to clozapine and sarcosine treatments.

Glycine N-methyltransferase (GNMT) affects cellular methylation capacity through regulating the ratio between S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). The product of its enzymatic reaction-sarcosine has antipsychotic effect in patients with schizophrenia. In this study, through RT-PCR and immunohistochemical staining, we demonstrated that GNMT expressed in various neurons located in the cerebral cortex, hippocampus, substantia nigra and cerebellum. Compared to the wild-type mice, Gnmt-/- mice had significantly lower level of sarcosine in the cerebral cortex. Real-time PCR identified genes involved in the methionine metabolism (Dnmt1 and Dnmt3a), ErbB (Nrg1 and ErbB4) and mTOR (Akt2, S6, S6k1 and S6k2) signaling pathways were dysregulated significantly in the cortex of Gnmt-/- mice. Acoustic startle reflex test demonstrated that Gnmt-/- mice had significantly lower level of prepulse inhibition and the deficit was ameliorated through clozapine or sarcosine treatment. Furthermore, liver-specific-human-GNMT transgenic with Gnmt-/- (Tg-GNMT/Gnmt-/-) mice were used to rule out that the phenotype was due to abnormal liver function. In summary, the neuropsychological abnormalities found in Gnmt-/- mice may represent an endophenotype of schizophrenia. GNMT plays an important role in maintaining normal physiological function of brain and Tg-GNMT/Gnmt-/- mice are useful models for development of therapeutics for patients with schizophrenia.

Increased Mauthner cell activity and escaping behaviour in seabream fed long-chain PUFA.

There is limited information on the specific effects of long-chain PUFA (LCPUFA) on neuron development and functioning. Deficiency of those essential fatty acids impairs escape and avoidance behaviour in fish, where Mauthner cells (M-cells) play a particularly important role in initiating this response. Gilthead seabream larvae fed two different LCPUFA profiles were challenged with a sonorous stimulus. Feeding n-3 LCPUFA increased the content of these fatty acids in fish tissues and caused a higher number of larvae to react to the stimulus with a faster burst swimming speed response. This faster startle response in fish fed n-3 LCPUFA was also associated with an increased immune-positive neural response, particularly in M-cells, denoting a higher production of acetylcholine. The present study shows the first evidence of the effect of n-3 LCPUFA on the functioning of particular neurons in fish, the M-cells and the behaviour response that they modulate to escape from a sound stimulus.

Otx2 and Otx1 protect diencephalon and mesencephalon from caudalization into metencephalon during early brain regionalization.

Otx2 is expressed in each step and site of head development. To dissect each Otx2 function we have identified a series of Otx2 enhancers. The Otx2 expression in the anterior neuroectoderm is regulated by the AN enhancer and the subsequent expression in forebrain and midbrain later than E8.5 by FM1 and FM2 enhancers; the Otx1 expression takes place at E8.0. In telencephalon later than E9.5 Otx1 continues to be expressed in the entire pallium, while the Otx2 expression is confined to the most medial pallium. To determine the Otx functions in forebrain and midbrain development we have generated mouse mutants that lack both FM1 and FM2 enhancers (DKO: Otx2(ΔFM1ΔFM2/ΔFM1ΔFM2)) and examined the TKO (Otx1(-/-)Otx2(ΔFM1ΔFM2/ΔFM1ΔFM2)) phenotype. The mutants develop normally until E8.0, but subsequently by E9.5 the diencephalon, including thalamic eminence and prethalamus, and the mesencephalon are caudalized into metencephalon consisting of isthmus and rhombomere 1; the caudalization does not extend to rhombomere 2 and more caudal rhombomeres. In rostral forebrain, neopallium, ganglionic eminences and hypothalamus in front of prethalamus develop; we propose that they become insensitive to the caudalization with the switch from the Otx2 expression under the AN enhancer to that under FM1 and FM2 enhancers. In contrast, the medial pallium requires Otx1 and Otx2 for its development later than E9.5, and the Otx2 expression in diencepalon and mesencephalon later than E9.5 is also directed by an enhancer other than FM1 and FM2 enhancers.

Mecanismos de compensación en la enfermedad de Parkinson / No disponible

No disponible

Neuroanatomy of the calf brain as revealed by high-resolution magnetic resonance imaging.

Here, we want to assess the benefit of high-resolution and high-contrast magnetic resonance imaging (MRI) for detailed documentation of internal brain morphology in formalin-fixed whole head specimens of the full-term calf brain (Bos taurus). Imaging was performed on a Siemens 1.5 T scanner. Optimum contrast was achieved using a 3D sequence with a flip angle of 30 degrees , repetition time (TR) of 20 ms, echo time (TE) of 6.8 ms, and an interpolated matrix of 1024 x 1024. In plane resolution was 0.25 mm. Computer-generated three-dimensional images were reconstructed from the original scans in the coronal plane. This study shows that MRI is capable to identify delicate structures in immature brain specimens. The use of MRI in comparative morphology facilitates the examination of series of brains or brain samples in a reasonable time. The comprehensive description of species- and group-specific brain features in MRI scans of Bos taurus will complement existing data for diagnostic imaging and neuromorphological research, in general, as well as for phylogenetic reconstructions.

Isthmus organizer for mesencephalon and metencephalon.

The vertebrate central nervous system is elaborated from a simple neural tube. Brain vesicles formation is the first sign of regionalization. Classical transplantation using quail and chick embryos revealed that the mesencephalon-metencephalon boundary (isthmus) functions as an organizer of the mesencephalon and metencephalon. Fgf8 is accepted as a main organizing molecule of the isthmus. Strong Fgf8 signal activates the Ras-ERK signaling pathway to differentiate the cerebellum. In this review, the historical background of the means of identifying the isthmus organizer and the molecular mechanisms of signal transduction for tectum and cerebellum differentiation is reviewed.

Lesion profiles and gliosis in the brainstem of 135 Swiss cows with bovine spongiform encephalopathy (BSE).

Lesion profiles are considered to be an important tool for the comparison of the various animal and human spongiform encephalopathies and to obtain information upon prion strain variations. Histological and immunohistochemical reactions (PrPsc, GFAP) in 13 brain areas at 4 levels in the brainstem from 135 BSE-positive and 45 BSE-negative cases were retrospectively evaluated. In this retrospective study a lesion profile based on histological features was worked out on the basis of BSE cases originating from Switzerland over a period of ten years. They were confirmed post mortem by histology and immunohistology. Our findings were reviewed in comparison with lesion profiles published in England. No striking differences comparing type and quality of lesions in the relevant areas between the Swiss and the English cases were evident. Moreover, the lesion profiles and the character of the lesions did not differ between animals born before or after the offal feeding ban, which supports the hypothesis that the Swiss epidemic is sustained by the same single, stable strain of the BSE agent, which is probably the same as in the English epidemic. There was a good correlation between PrPsc accumulation and spongiform changes, in particular in those areas which were morphologically most affected. Astrocytosis in BSE was quantified. A significant rise in GFAP-positive cells could be shown comparing the brain stem nuclei of BSE affected with BSE-unaffected cattle, despite considerable variation between the cases and between the nuclei. The observed astrocytosis did correlate with vacuolation of the neuropil and of perikarya as well as with PrPsc accumulation.

Effect of the administration of tiagabine and gabapentin on rabbit electroencephalogram activity.

New generation antiepileptic drugs, including gabapentin and tiagabine, are used in monotherapy or in combination with other drugs for specific seizure types. The multidirectional mechanism of activity and varied pharmacological properties of these drugs suggest that they could also be used in the therapy of other diseases. A possible limitation of new generation antiepileptic drugs is the incidence of CNS-related adverse effects. Few studies have assessed the effect of new antiepileptic drugs on electroencephalogram (EEG) recordings in subjects using these drugs for diseases other than epilepsy. The aim of this study was to determine the effects of tiagabine and gabapentin on EEG recordings from the midbrain reticular formation, dorsal hippocampus and frontal cortex in rabbits. Tiagabine was administered orally at a single dose of 5 and 20 mg kg(-1), or repeatedly at a dose of 5 mg kg(-1) (twice a day) for 14 days. Gabapentin was administered orally at a single dose of 25 and 100 mg kg(-1), or repeatedly at a dose of 25 mg kg(-1) (twice a day) for 14 days. Both tiagabine and gabapentin caused changes indicative of CNS inhibitory properties, which may be associated with the adverse effects of the drugs. After repeated doses of the drugs, the changes in EEG recordings were less pronounced than after single doses, which may indicate adaptive changes. The hippocampus was found to be the least sensitive to the effect of gabapentin.

Analysis and visualization of cell movement in the developing zebrafish brain.

Detailed reconstruction of the spatiotemporal history of embryonic cells is key to understanding tissue formation processes but is often complicated by the large number of cells involved, particularly so in vertebrates. Through a combination of high-resolution time-lapse lineage tracing and antibody staining, we have analyzed the movement of mesencephalic and metencephalic cell populations in the early zebrafish embryo. To facilitate the analysis of our cell tracking data, we have created TracePilot, a software tool that allows interactive manipulation and visualization of tracking data. We demonstrate its utility by showing novel visualizations of cell movement in the developing zebrafish brain. TracePilot (http://www.mpi-cbg.de/tracepilot) is Java-based, available free of charge, and has a program structure that allows the incorporation of additional analysis tools.

Zebrafish Lmx1b.1 and Lmx1b.2 are required for maintenance of the isthmic organizer.

The mesencephalic and metencephalic region (MMR) of the vertebrate central nervous system develops in response to signals produced by the isthmic organizer (IsO). We have previously reported that the LIM homeobox transcription factor Lmx1b is expressed within the chick IsO, where it is sufficient to maintain expression of the secreted factor wnt1. In this paper, we show that zebrafish express two Lmx1b orthologs, lmx1b.1 and lmx1b.2, in the rostral IsO, and demonstrate that these genes are necessary for key aspects of MMR development. Simultaneous knockdown of Lmx1b.1 and Lmx1b.2 using morpholino antisense oligos results in a loss of wnt1, wnt3a, wnt10b, pax8 and fgf8 expression at the IsO, leading ultimately to programmed cell death and the loss of the isthmic constriction and cerebellum. Single morpholino knockdown of either Lmx1b.1 or Lmx1b.2 has no discernible effect on MMR development. Maintenance of lmx1b.1 and lmx1b.2 expression at the isthmus requires the function of no isthmus/pax2.1, as well as Fgf signaling. Transient misexpression of Lmx1b.1 or Lmx1b.2 during early MMR development induces ectopic wnt1 and fgf8 expression in the MMR, as well as throughout much of the embryo. We propose that Lmx1b.1- and Lmx1b.2-mediated regulation of wnt1, wnt3a, wnt10b, pax8 and fgf8 maintains cell survival in the isthmocerebellar region.

Isthmus organizer and regionalization of the mesencephalon and metencephalon.

The brain vesicles that are formed at an early stage of neural development are the fundamentals of the brain plan. Heterotopic transplantation revealed that the diencephalon could change its fate when juxtaposed to the isthmus (mes-metencephalic boundary), which indicated that the isthmus functions as an organizer for the mesencephalon and metencephalon. Fgf8 is identified as an isthmus organizing signal. Misexpression of Fgf8a and Fgf8b indicated that a strong Fgf8 signal organizes cerebellar development. The transcription factors define the fate of the region. Overlapping expression of Otx2, En1 and Pax2 may define the mesencephalic region and additional expression of Pax3/7 may instruct the mesencephalic region to differentiate into the tectum. The di-mesencephalic boundary is determined by repressive interaction between Pax6 and En1/Pax2 and the mes-metencephalic boundary is defined by repressive interaction between Otx2 and Gbx2. Fgf8 is induced at the border of the Otx2 and Gbx2 expression domain, overlapping with Gbx2 expression.