Deficits in cerebellar granule cell development and social interactions in CD47 knockout mice

CD47 is involved in neurite differentiation in cultured neurons, but the function of CD47 in brain development is largely unknown. We determined that CD47 mRNA was robustly expressed in the developing cerebellum, especially in granule cells. CD47 protein was mainly expressed in the inner layer of the external granule layer (EGL), molecular layer, and internal granule layer (IGL), where granule cells individually become postmitotic and migrate, leading to neurite fasciculation. At postnatal day 8 (P8), CD47 knockout mice exhibited an increased number of proliferating granule cells in the EGL, whereas the CD47 agonist peptide 4N1K increased the number of postmitotic cells in primary granule cells. Knocking out the CD47 gene and anti‐CD47 antibody impaired the radial migration of granule cells from the EGL to the IGL individually in mice and slice cultures. In primary granule cells, knocking out CD47 reduced the number of axonal collaterals and dendritic branches; by contrast, overexpressing CD47 or 4N1K treatment increased the axonal length and numbers of axonal collaterals and dendritic branches. Furthermore, the length of the fissure between Lobules VI and VII was decreased in CD47 knockout mice at P21 and at 14 wk after birth. Lastly, CD47 knockout mice exhibited increased social interaction at P21 and depressive‐like behaviors at 10 wk after birth. Our study revealed that the cell adhesion molecule CD47 participates in multiple phases of granule cell development, including proliferation, migration, and neurite differentiation implying that aberrations of CD47 are risk factors that cause abnormalities in cerebellar development and atypical behaviors.© 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 463–484, 2015     

Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells

Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. We examined the roles of ryanodine receptor (RyR), an intracellular Ca²⁺ release channel, in the dendrite formation of Purkinje cells using cerebellar cell cultures. In the cerebellum, Purkinje cells express RyR1 and RyR2, whereas granule cells express RyR2. When ryanodine (10 µM), a blocker of RyR, was added to the culture medium, the elongation and branching of Purkinje cell dendrites were markedly inhibited. When we transferred small interfering RNA (siRNA) against RyR1 into Purkinje cells using single‐cell electroporation, dendritic branching but not elongation of the electroporated Purkinje cells was inhibited. On the other hand, transfection of RyR2 siRNA into granule cells also inhibited dendritic branching of Purkinje cells. Furthermore, ryanodine reduced the levels of brain‐derived neurotrophic factor (BDNF) in the culture medium. The ryanodine‐induced inhibition of dendritic differentiation was partially rescued when BDNF was exogenously added to the culture medium in addition to ryanodine. Overall, these results suggest that RyRs expressed by both Purkinje and granule cells play important roles in promoting the dendritic differentiation of Purkinje cells and that RyR2 expressed by granule cells is involved in the secretion of BDNF from granule cells. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 467–480, 2014     

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.     

Endogenous PACAP acts as a stress response peptide to protect cerebellar neurons from ethanol or oxidative insult

The rodent cerebellum is richly supplied with PACAPergic innervation. Exogenous pituitary adenylate cyclase-activating polypeptide (PACAP) increases cerebellar granule cell survival and differentiation in culture, and enhances the number of neuroblasts in the molecular and internal granule cell layers (IGL) when injected postnatally into the cerebellum in vivo. Here, we have investigated the role of endogenous PACAP during cerebellar development by comparing the morphology of normal and PACAP-deficient mouse cerebellum, and the response of cerebellar granule cells from normal and PACAP-deficient mice subjected to neurotoxic insult in culture. There was no difference in cerebellar volume or granule cell number, in 11-day-old wild type versus PACAP-deficient mice. Cultured cerebellar neurons from PACAP-deficient and wild type mice also showed no apparent differences in survival and differentiation either under depolarizing conditions, or non-depolarizing conditions in the presence or absence of either dibutyryl cAMP or 100 nM PACAP. However, cultured cerebellar neurons from PACAP-deficient mice were significantly more sensitive than wild type neurons to ethanol- or hydrogen peroxide-induced toxicity. Differential ethanol toxicity was reversed by addition of 100 nM exogenous PACAP, suggesting that endogenous PACAP has neuroprotective activity in the context of cellular insult or stress. The neuroprotective action of PACAP was mimicked by dibutryl cAMP, indicating that it occurred via activation of adenylate cyclase. These results indicate that PACAP might act to protect the brain from paraphysiological insult, including exposure to toxins or hypoxia.     

The zebrafish cerebellar rhombic lip is spatially patterned in producing granule cell populations of different functional compartments

The upper rhombic lip, a prominent germinal zone of the cerebellum, was recently demonstrated to generate different neuronal cell types over time from spatial subdomains. We have characterized the differentiation of the upper rhombic lip derived granule cell population in stable GFP-transgenic zebrafish in the context of zebrafish cerebellar morphogenesis. Time-lapse analysis followed by individual granule cell tracing demonstrates that the zebrafish upper rhombic lip is spatially patterned along its mediolateral axis producing different granule cell populations simultaneously. Time-lapse recordings of parallel fiber projections and retrograde labeling reveal that spatial patterning within the rhombic lip corresponds to granule cells of two different functional compartments of the mature cerebellum: the eminentia granularis and the corpus cerebelli. These cerebellar compartments in teleosts correspond to the mammalian vestibulocerebellar and non-vestibulocerebellar system serving balance and locomotion control, respectively. Given the high conservation of cerebellar development in vertebrates, spatial partitioning of the mammalian granule cell population and their corresponding earlier-produced deep nuclei by patterning within the rhombic lip may also delineate distinct functional compartments of the cerebellum. Thus, our findings offer an explanation for how specific functional cerebellar circuitries are laid down by spatio-temporal patterning of cerebellar germinal zones during early brain development.     

Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation [published erratum appears in J Cell Biol 1998 Dec 28;143(7):following 2090]

Thyroid hormones play an important role in brain development, but the mechanism(s) by which triiodothyronine (T3) mediates neuronal differentiation is poorly understood. Here we demonstrate that T3 regulates the neurotrophic factor, neurotrophin-3 (NT-3), in developing rat cerebellar granule cells both in cell culture and in vivo. In situ hybridization experiments showed that developing Purkinje cells do not express NT-3 mRNA but do express trkC, the putative neuronal receptor for NT-3. Addition of recombinant NT-3 to cerebellar cultures from embryonic rat brain induces hypertrophy and neurite sprouting of Purkinje cells, and upregulates the mRNA encoding the calcium-binding protein, calbindin-28 kD. The present study demonstrates a novel interaction between cerebellar granule neurons and developing Purkinje cells in which NT-3 induced by T3 in the granule cells promotes Purkinje cell differentiation.     

Oligodendrocyte ablation impairs cerebellum development

Oligodendrocytes (OLs) are the glial cells of the central nervous system and are classically known to form myelin sheaths around most axons of higher vertebrates. Whether these cells might have other roles, in particular during development, has not been studied. Taking advantage of a transgenic mouse model in which OLs can be selectively killed in a desired time-frame, we have investigated the impact of OL ablation on cerebellar development. OL ablation was induced during the first 3 postnatal weeks, a time at which cerebellum development is ongoing. Strikingly, OL ablation triggers a profound perturbation of the known cerebellum developmental program, characterized by the disorganization of the cortical layers, abnormal foliation and a complete alteration of Purkinje cell dendritic arborization and axonal fasciculation. This phenotype is accompanied by decreased granule cell density, a disorganized Bergmann glia network and impaired migration of interneurons in the molecular layer. These results demonstrate a previously ignored role of OLs in the formation of the cerebellar cytoarchitecture.     

WNT signaling increases proliferation and impairs differentiation of stem cells in the developing cerebellum

The WNT pathway plays multiple roles in neural development and is crucial for establishment of the embryonic cerebellum. In addition, WNT pathway mutations are associated with medulloblastoma, the most common malignant brain tumor in children. However, the cell types within the cerebellum that are responsive to WNT signaling remain unknown. Here we investigate the effects of canonical WNT signaling on two important classes of progenitors in the developing cerebellum: multipotent neural stem cells (NSCs) and granule neuron precursors (GNPs). We show that WNT pathway activation in vitro promotes proliferation of NSCs but not GNPs. Moreover, mice that express activated β-catenin in the cerebellar ventricular zone exhibit increased proliferation of NSCs in that region, whereas expression of the same protein in GNPs impairs proliferation. Although β-catenin-expressing NSCs proliferate they do not undergo prolonged expansion or neoplastic growth; rather, WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level, mutant NSCs exhibit increased expression of c-Myc, which might account for their transient proliferation, but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21, which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation.     

Transgenic mice expressing F3/contactin from the TAG-1 promoter exhibit developmentally regulated changes in the differentiation of cerebellar neurons

F3/contactin (CNTN1) and TAG-1 (CNTN2) are closely related axonal glycoproteins that are differentially regulated during development. In the cerebellar cortex TAG-1 is expressed first as granule cell progenitors differentiate in the premigratory zone of the external germinal layer. However, as these cells begin radial migration, TAG-1 is replaced by F3/contactin. To address the significance of this differential regulation, we have generated transgenic mice in which F3/contactin expression is driven by TAG-1 gene regulatory sequences, which results in premature expression of F3/contactin in granule cells. These animals (TAG/F3 mice) display a developmentally regulated cerebellar phenotype in which the size of the cerebellum is markedly reduced during the first two postnatal weeks but subsequently recovers. This is due in part to a reduction in the number of granule cells, most evident in the external germinal layer at postnatal day 3 and in the inner granular layer between postnatal days 8 and 11. The reduction in granule cell number is accompanied by a decrease in precursor granule cell proliferation at postnatal day 3, followed by an increase in the number of cycling cells at postnatal day 8. In the same developmental window the size of the molecular layer is markedly reduced and Purkinje cell dendrites fail to elaborate normally. These data are consistent with a model in which deployment of F3/contactin on granule cells affects proliferation and differentiation of these neurons as well as the differentiation of their synaptic partners, the Purkinje cells. Together, these findings indicate that precise spatio-temporal regulation of TAG-1 and F3/contactin expression is critical for normal cerebellar morphogenesis.     

Mouse Model Reveals the Role of RERE in Cerebellar Foliation and the Migration and Maturation of Purkinje Cells

Nuclear receptors and their coregulators play a critical role in brain development by regulating the spatiotemporal expression of their target genes. The arginine-glutamic acid dipeptide repeats gene (Rere) encodes a nuclear receptor coregulator previously known as Atrophin 2. In the developing cerebellum, RERE is expressed in the molecular layer, the Purkinje cell layer and the granule cell layer but not in granule cell precursors. To study RERE''s role in cerebellar development, we used RERE-deficient embryos bearing a null allele (om) and a hypomorphic allele (eyes3) of Rere (Rere ^om/eyes3). In contrast to wild-type embryos, formation of the principal fissures in these RERE-deficient embryos was delayed and the proliferative activity of granule cell precursors (GCPs) was reduced at E18.5. This reduction in proliferation was accompanied by a decrease in the expression of sonic hedgehog (SHH), which is secreted from Purkinje cells and is required for normal GCP proliferation. The maturation and migration of Purkinje cells in Rere ^om/eyes3 embryos was also delayed with decreased numbers of post-migratory Purkinje cells in the cerebellum. During the postnatal period, RERE depletion caused incomplete division of lobules I/II and III due to truncated development of the precentral fissure in the cerebellar vermis, abnormal development of lobule crus I and lobule crus II in the cerebellar hemispheres due to attenuation of the intercrural fissure, and decreased levels of Purkinje cell dendritic branching. We conclude that RERE-deficiency leads to delayed development of the principal fissures and delayed maturation and migration of Purkinje cells during prenatal cerebellar development and abnormal cerebellar foliation and Purkinje cell maturation during postnatal cerebellar development.     

The calcium‐sensing receptor and integrins modulate cerebellar granule cell precursor differentiation and migration

In the developing cerebellum granule cell precursors (GCPs) proliferate in the external granule cell layer before differentiating and migrating to the inner granule cell layer. Aberrant GCP proliferation leads to medulloblastoma, the most prevalent form of childhood brain cancer. Here, we demonstrate that the calcium‐sensing receptor (CaSR), a homodimeric G‐protein coupled receptor, functions in conjunction with cell adhesion proteins, the integrins, to enhance GCP migration and cell homing by promoting GCP differentiation. During the second postnatal week a robust peak in CaSR expression was observed in GCPs; reciprocal immunoprecipitation experiments conducted during this period established that the CaSR and β1 integrins are present together in a macromolecular protein complex. Analysis of cell‐surface proteins demonstrated that activation of the CaSR by positive allosteric modulators promoted plasma membrane expression of β1 integrins via ERK2 and AKT phosphorylation and resulted in increased GCP migration toward an extracellular matrix protein. The results of in vivo experiments whereby CaSR modulators were injected i.c.v. revealed that CaSR activation promoted radial migration of GCPs by enhancing GCP differentiation, and conversely, a CaSR inhibitor disrupted GCP differentiation and promoted GCP proliferation. Our results demonstrate that an ion‐sensing G‐protein coupled receptor acts to promote neuronal differentiation and homing during cerebellar maturation. These findings together with those of others also suggest that CaSR/integrin complexes act to transduce extracellular calcium signals into cellular movement, and may function in this capacity as a universal cell migration/homing complex in the developing brain. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 375–389, 2016     

Development of polarity in cerebellar granule neurons

Axon formation in developing cerebellar granule neurons in situ is spatially and temporally segregated from subsequent neuronal migration and dendrite formation. To examine the role of local environmental cues on early steps in granule cell differentiation, the sequence of morphologic development and polarized distribution of membrane proteins was determined in granule cells isolated from contact with other cerebellar cell types. Granule cells cultured at low density developed their characteristic axonal and dendritic morphologies in a series of discrete temporal steps highly similar to those observed in situ, first extending a unipolar process, then long, thin bipolar axons, and finally becoming multipolar, forming short dendrites around the cell body. Axonal- and dendritic-specific cytoskeletal markers were segregated to the morphologically distinct domains. The cell surface distribution of a specific class of endogenous glycoproteins, those linked to the membrane by a glycosylphosphatidyl inositol (GPI) anchor, was also examined. The GPI-anchored protein, TAG-1, which is segregated to the parallel fiber axons in situ, was found exclusively on granule cell axons in vitro; however, two other endogenous GPI-anchored proteins were found on both the axonal and somatodendritic domains. These results demonstrate that granule cells develop polarity in a cell type-specific manner in the absence of the spatial cues of the developing cerebellar cortex. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 223–236, 1997.     

Shp2 acts downstream of SDF-1α/CXCR4 in guiding granule cell migration during cerebellar development

Shp2 is a non-receptor protein tyrosine phosphatase containing two Src homology 2 (SH2) domains that is implicated in intracellular signaling events controlling cell proliferation, differentiation and migration. To examine the role of Shp2 in brain development, we created mice with Shp2 selectively deleted in neural stem/progenitor cells. Homozygous mutant mice exhibited early postnatal lethality with defects in neural stem cell self-renewal and neuronal/glial cell fate specification. Here we report a critical role of Shp2 in guiding neuronal cell migration in the cerebellum. In homozygous mutants, we observed reduced and less foliated cerebellum, ectopic presence of external granule cells and mispositioned Purkinje cells, a phenotype very similar to that of mutant mice lacking either SDF-1α or CXCR4. Consistently, Shp2-deficient granule cells failed to migrate toward SDF-1α in an in vitro cell migration assay, and SDF-1α treatment triggered a robust induction of tyrosyl phosphorylation on Shp2. Together, these results suggest that although Shp2 is involved in multiple signaling events during brain development, a prominent role of the phosphatase is to mediate SDF-1α/CXCR4 signal in guiding cerebellar granule cell migration.     

Impaired Cerebellar Development in Mice Overexpressing VGF

VGF nerve growth factor inducible (VGF) is a neuropeptide precursor induced by brain-derived neurotrophic factor and nerve growth factor. VGF is increased in the prefrontal cortex and cerebrospinal fluid in schizophrenia patients. In our previous study, VGF-overexpressing mice exhibited schizophrenia-like behaviors and smaller brain weights. Brain developmental abnormality is one cause of mental illness. Research on brain development is important for discovery of pathogenesis of mental disorders. In the present study, we investigated the role of VGF on cerebellar development. We performed a histological analysis with cerebellar sections of adult and postnatal day 3 mice by Nissl staining. To investigate cerebellar development, we performed immunostaining with antibodies of immature and mature granule cell markers. To understand the mechanism underlying these histological changes, we examined MAPK, Wnt, and sonic hedgehog signaling by Western blot. Finally, we performed rotarod and footprint tests using adult mice to investigate motor function. VGF-overexpressing adult mice exhibited smaller cerebellar sagittal section area. In postnatal day 3 mice, a cerebellar sagittal section area reduction of the whole cerebellum and external granule layer and a decrease in the number of mature granule cells were found in VGF-overexpressing mice. Additionally, the number of proliferative granule cell precursors was lower in VGF-overexpressing mice. Phosphorylation of Trk and Erk1 were increased in the cerebellum of postnatal day 3 VGF-overexpressing mice. Adult VGF-overexpressing mice exhibited motor disability. All together, these findings implicate VGF in the development of cerebellar granule cells via promoting MAPK signaling and motor function in the adult stage.

     

Sonic hedgehog regulates the growth and patterning of the cerebellum.

The molecular bases of brain development and CNS malignancies remain poorly understood. Here we show that Sonic hedgehog (Shh) signaling controls the development of the cerebellum at multiple levels. SHH is produced by Purkinje neurons, it is required for the proliferation of granule neuron precursors and it induces the differentiation of Bergmann glia. Blocking SHH function in vivo results in deficient granule neuron and Bergmann glia differentiation as well as in abnormal Purkinje neuron development. Thus, our findings provide a molecular model for the growth and patterning of the cerebellum by SHH through the coordination of the development of cortical cerebellar cell types. In addition, they provide a cellular context for medulloblastomas, childhood cancers of the cerebellum.     

A Novel Zinc Finger Protein, Zic, Is Involved in Neurogenesis, Especially in the Cell Lineage of Cerebellar Granule Cells

Abstract: To clarify the mechanism of cerebellar development, we have cloned a gene, named zic, encoding a zinc finger protein that is expressed abundantly in granule cells throughout development of the cerebellum. zic has a significant homology to the zinc finger domain of the Caenorhabditis elegans tra1 gene, the Drosophila cubitus interruptus Dominant gene, and the human GLI oncogene. An in situ hybridization study revealed that zic showed a restricted expression pattern in the granule cells and their putative precursor cells. It is also expressed at an early embryonic stage in the dorsal half of the neural tube. The expression pattern and nuclear localization were confirmed by immunohistochemical study. Furthermore, the bacterially expressed zic protein containing the zinc finger domains bound to the GLI -binding sequence. These findings suggest that zic is one of a number of nuclear factors involved in both differentiation in early development and maintenance of properties of the cerebellar granule cells.     

α-internexin is the only neuronal intermediate filament expressed in developing cerebellar granule neurons

We have used immunocytochemistry and in situ hybridization to examine the distribution of neuronal intermediate filament proteins and their mRNAs in the developing mouse cerebellum. First, we demonstrate that α-internexin is abundantly expressed in the developing cerebellum and is the only neuronal intermediate filament protein expressed in developing, including migrating, granule neurons. Second, in granule neuron reaggregates in vitro, α-internexin is the only neuronal intermediate filament protein highly expressed in the processes of the cultured granule neurons. This in vitro observation is consistent with results from immunocytochemistry and in situ hybridization studies of developing granule neurons in vivo, which suggest that α-internexin is the major neuronal intermediate filament protein in developing granule neurons. Finally, the neurofilament triplet proteins are expressed later, and coexist with α-internexin in other cells, including Purkinje cells and interneurons in the mature mouse cerebellum. These changes in neuronal intermediate filament composition may regulate neuronal maturation and axonal stability in cerebellar development. Furthermore, α-internexin may play a key role in neurite outgrowth and the establishment of neuronal cytoarchitecture. © 1996 John Wiley & Sons, Inc.     

Axonal Membrane-Skeletal Protein A60: Association with a Brain Spectrin-Binding Activity and Entry into Cerebellar Axons at a Stage After the Initiation of Axonal Growth

Abstract: A60 is a 60-kDa component of the axonal cortical cytoskeleton in CNS neurones. It appears to be neurone specific and is tightly bound to brain membranes. In this study the cytoskeletal activities and developmental expression of A60 in rat cerebellum have been examined using the monoclonal antibody DR1. A60 in a partially purified soluble extract of brain membranes interacts selectively with brain but not erythrocyte spectrin. Because erythrocyte spectrin is more closely related to the dendritic form of spectrin than the axonal form, this raises the possibility that AGO localises in axons by interaction with the axonal form of spectrin only. A60 is not found in rat cerebellum before the day of birth. However, during postnatal development of the cerebellum (days 1–13) DR1 reactivity appears progressively. On postnatal day 1, a small population of cells in the mantle layer (presumptive Purkinje cells) is DR1 positive. There is no DR1 reactivity found in Purkinje cell axons during their initial phase of growth. By postnatal day 7, Purkinje cell bodies, initial dendritic segments, and the cerebellar white matter are all positive. This pattern of labelling is strengthened up until postnatal day 13. By contrast, in adult rat cerebellum, the location of A60 has changed so that it is most concentrated in axons, and dendritic staining is lost. These data indicate that A60 is a spectrin-binding component of the adult axonal membrane skeleton, the presence of which is only required in axons after the initial phase of growth.