Purkinje Cell Differentiation Research Articles

Foxp and Skor family proteins control differentiation of Purkinje cells from Ptf1a- and Neurog1-expressing progenitors in zebrafish.

Cerebellar neurons, such as GABAergic Purkinje cells (PCs), interneurons (INs) and glutamatergic granule cells (GCs) are differentiated from neural progenitors expressing proneural genes, including ptf1a, neurog1 and atoh1a/b/c. Studies in mammals previously suggested that these genes determine cerebellar neuron cell fate. However, our studies on ptf1a;neurog1 zebrafish mutants and lineage tracing of ptf1a-expressing progenitors have revealed that the ptf1a/neurog1-expressing progenitors can generate diverse cerebellar neurons, including PCs, INs and a subset of GCs in zebrafish. The precise mechanisms of how each cerebellar neuron type is specified remains elusive. We found that genes encoding the transcriptional regulators Foxp1b, Foxp4, Skor1b and Skor2, which are reportedly expressed in PCs, were absent in ptf1a;neurog1 mutants. foxp1b;foxp4 mutants showed a strong reduction in PCs, whereas skor1b;skor2 mutants completely lacked PCs, and displayed an increase in immature GCs. Misexpression of skor2 in GC progenitors expressing atoh1c suppressed GC fate. These data indicate that Foxp1b/4 and Skor1b/2 function as key transcriptional regulators in the initial step of PC differentiation from ptf1a/neurog1-expressing neural progenitors, and that Skor1b and Skor2 control PC differentiation by suppressing their differentiation into GCs.

Functional regionalization of the differentiating cerebellar Purkinje cell population occurs in an activity-dependent manner.

The cerebellum is organized into functional regions each dedicated to process different motor or sensory inputs for controlling different locomotor behaviors. This functional regionalization is prominent in the evolutionary conserved single-cell layered Purkinje cell (PC) population. Fragmented gene expression domains suggest a genetic organization of PC layer regionalization during cerebellum development. However, the establishment of such functionally specific domains during PC differentiation remained elusive. We show the progressive emergence of functional regionalization of PCs from broad responses to spatially restricted regions in zebrafish by means of in vivo Ca2+-imaging during stereotypic locomotive behavior. Moreover, we reveal that formation of new dendritic spines during cerebellar development using in vivo imaging parallels the time course of functional domain development. Pharmacological as well as cell-type specific optogenetic inhibition of PC neuronal activity results in reduced PC dendritic spine density and an altered stagnant pattern of functional domain formation in the PC layer. Hence, our study suggests that functional regionalization of the PC layer is driven by physiological activity of maturing PCs themselves.

Lobule-Related Action Potential Shape- and History-Dependent Current Integration in Purkinje Cells of Adult and Developing Mice.

Purkinje cells (PCs) are the principal cells of the cerebellar cortex and form a central element in the modular organization of the cerebellum. Differentiation of PCs based on gene expression profiles revealed two subpopulations with distinct connectivity, action potential firing and learning-induced activity changes. However, which basal cell physiological features underlie the differences between these subpopulations and to what extent they integrate input differentially remains largely unclear. Here, we investigate the cellular electrophysiological properties of PC subpopulation in adult and juvenile mice. We found that multiple fundamental cell physiological properties, including membrane resistance and various aspects of the action potential shape, differ between PCs from anterior and nodular lobules. Moreover, the two PC subpopulations also differed in the integration of negative and positive current steps as well as in size of the hyperpolarization-activated current. A comparative analysis in juvenile mice confirmed that most of these lobule-specific differences are already present at pre-weaning ages. Finally, we found that current integration in PCs is input history-dependent for both positive and negative currents, but this is not a distinctive feature between anterior and nodular PCs. Our results support the concept of a fundamental differentiation of PCs subpopulations in terms of cell physiological properties and current integration, yet reveals that history-dependent input processing is consistent across PC subtypes.

PTRH2 is Necessary for Purkinje Cell Differentiation and Survival and its Loss Recapitulates Progressive Cerebellar Atrophy and Ataxia Seen in IMNEPD Patients

Homozygous variants in the peptidyl-tRNA hydrolase 2 gene (PTRH2) cause infantile-onset multisystem neurologic, endocrine, and pancreatic disease. The objective is to delineate the mechanisms underlying the core cerebellar phenotype in this disease. For this, we generated constitutive (Ptrh2LoxPxhCMVCre, Ptrh2−/− mice) and Purkinje cell (PC) specific (Ptrh2LoxPxPcp2Cre, Ptrh2ΔPCmice) Ptrh2 mutant mouse models and investigated the effect of the loss of Ptrh2 on cerebellar development. We show that Ptrh2−/− knockout mice had severe postnatal runting and lethality by postnatal day 14. Ptrh2ΔPC PC specific knockout mice survived until adult age; however, they showed progressive cerebellar atrophy and functional cerebellar deficits with abnormal gait and ataxia. PCs of Ptrh2ΔPC mice had reduced cell size and density, stunted dendrites, and lower levels of ribosomal protein S6, a readout of the mammalian target of rapamycin pathway. By adulthood, there was a marked loss of PCs. Thus, we identify a cell autonomous requirement for PTRH2 in PC maturation and survival. Loss of PTRH2 in PCs leads to downregulation of the mTOR pathway and PC atrophy. This suggests a molecular mechanism underlying the ataxia and cerebellar atrophy seen in patients with PTRH2 mutations leading to infantile-onset multisystem neurologic, endocrine, and pancreatic disease.

Recessive PRDM13 mutations cause fatal perinatal brainstem dysfunction with cerebellar hypoplasia and disrupt Purkinje cell differentiation

Pontocerebellar hypoplasias (PCHs) are congenital disorders characterized by hypoplasia or early atrophy of the cerebellum and brainstem, leading to a very limited motor and cognitive development. Although over 20 genes have been shown to be mutated in PCHs, a large proportion of affected individuals remains undiagnosed. We describe four families with children presenting with severe neonatal brainstem dysfunction and pronounced deficits in cognitive and motor development associated with four different bi-allelic mutations in PRDM13, including homozygous truncating variants in the most severely affected individuals. Brain MRI and fetopathological examination revealed a PCH-like phenotype, associated with major hypoplasia of inferior olive nuclei and dysplasia of the dentate nucleus. Notably, histopathological examinations highlighted a sparse and disorganized Purkinje cell layer in the cerebellum. PRDM13 encodes a transcriptional repressor known to be critical for neuronal subtypes specification in the mouse retina and spinal cord but had not been implicated, so far, in hindbrain development. snRNA-seq data mining and in situ hybridization in humans show that PRDM13 is expressed at early stages in the progenitors of the cerebellar ventricular zone, which gives rise to cerebellar GABAergic neurons, including Purkinje cells. We also show that loss of function of prdm13 in zebrafish leads to a reduction in Purkinje cells numbers and a complete absence of the inferior olive nuclei. Altogether our data identified bi-allelic mutations in PRDM13 as causing a olivopontocerebellar hypoplasia syndrome and suggest that early deregulations of the transcriptional control of neuronal fate specification could contribute to a significant number of cases.

Olig3 regulates early cerebellar development.

The mature cerebellum controls motor skill precision and participates in other sophisticated brain functions that include learning, cognition, and speech. Different types of GABAergic and glutamatergic cerebellar neurons originate in temporal order from two progenitor niches, the ventricular zone and rhombic lip, which express the transcription factors Ptf1a and Atoh1, respectively. However, the molecular machinery required to specify the distinct neuronal types emanating from these progenitor zones is still unclear. Here, we uncover the transcription factor Olig3 as a major determinant in generating the earliest neuronal derivatives emanating from both progenitor zones in mice. In the rhombic lip, Olig3 regulates progenitor cell proliferation. In the ventricular zone, Olig3 safeguards Purkinje cell specification by curtailing the expression of Pax2, a transcription factor that suppresses the Purkinje cell differentiation program. Our work thus defines Olig3 as a key factor in early cerebellar development.

Promotion of Dendritic Differentiation of Cerebellar Purkinje Cells by Ca2+/calmodulin-dependent Protein Kinase IIα, IIβ and IV and Possible Involvement of CREB Phosphorylation

Cerebellar Purkinje cells develop the most elaborate dendritic trees among neurons in the brain. To examine the role of Ca2+/calmodulin-dependent protein kinase (CaMK) IIα, IIβ and IV in the dendritic differentiation of Purkinje cells, we introduced siRNA against these CaMKs into Purkinje cells in cerebellar cell cultures using a single-cell electroporation technique. Single-cell electroporation enables us to transfer siRNA into specific cells within a heterogeneous cell population. In addition, we can easily and reliably transfer multiple types of siRNA into a cell simply by loading them together in one micropipette. Any one of the siRNA against CaMKIIα, IIβ and IV (single knockdown) or any combinations of two of the siRNA against these CaMKs (double knockdown) had no significant effects on the dendritic differentiation of Purkinje cells. However, the combination of all three siRNA against these CaMKs (triple knockdown) inhibited the branching of Purkinje cell dendrites. Furthermore, the triple knockdown reduced the phosphorylation of CREB in Purkinje cells. These findings suggest the promotion of dendritic differentiation of Purkinje cells by CaMKIIα, IIβ and IV and the possible involvement of phosphorylation of CREB as a common substrate of these CaMKs.

Gpr37l1/prosaposin receptor regulates Ptch1 trafficking, Shh production, and cell proliferation in cerebellar primary astrocytes.

Mammalian cerebellar astrocytes critically regulate the differentiation and maturation of neuronal Purkinje cells and granule precursors. The G protein-coupled receptor 37-like 1 (Gpr37l1) is expressed by Bergmann astrocytes and interacts with patched 1 (Ptch1) at peri-ciliary membranes. Cerebellar primary astrocyte cultures from wild-type and Gpr37l1 null mutant mouse pups were established and studied. Primary cilia were produced by cultures of both genotypes, as well as Ptch1 and smoothened (Smo) components of the sonic hedgehog (Shh) mitogenic pathway. Compared to wild-type cells, Gpr37l1-/- astrocytes displayed striking increases in proliferative activity, Ptch1 protein expression and internalization, intracellular cholesterol content, ciliary localization of Smo, as well as a marked production of active Shh. Similar effects were reproduced by treating wild-type astrocytes with a putative prosaptide ligand of Gpr37l1. These findings indicate that Gpr37l1-Ptch1 interactions specifically regulate Ptch1 internalization and trafficking, with consequent stimulation of Shh production and activation of proliferative signaling.

Novel genetic features of human and mouse Purkinje cell differentiation defined by comparative transcriptomics

Comparative transcriptomics between differentiating human pluripotent stem cells (hPSCs) and developing mouse neurons offers a powerful approach to compare genetic and epigenetic pathways in human and mouse neurons. To analyze human Purkinje cell (PC) differentiation, we optimized a protocol to generate human pluripotent stem cell-derived Purkinje cells (hPSC-PCs) that formed synapses when cultured with mouse cerebellar glia and granule cells and fired large calcium currents, measured with the genetically encoded calcium indicator jRGECO1a. To directly compare global gene expression of hPSC-PCs with developing mouse PCs, we used translating ribosomal affinity purification (TRAP). As a first step, we used Tg(Pcp2-L10a-Egfp) TRAP mice to profile actively transcribed genes in developing postnatal mouse PCs and used metagene projection to identify the most salient patterns of PC gene expression over time. We then created a transgenic Pcp2-L10a-Egfp TRAP hPSC line to profile gene expression in differentiating hPSC-PCs, finding that the key gene expression pathways of differentiated hPSC-PCs most closely matched those of late juvenile mouse PCs (P21). Comparative bioinformatics identified classical PC gene signatures as well as novel mitochondrial and autophagy gene pathways during the differentiation of both mouse and human PCs. In addition, we identified genes expressed in hPSC-PCs but not mouse PCs and confirmed protein expression of a novel human PC gene, CD40LG, expressed in both hPSC-PCs and native human cerebellar tissue. This study therefore provides a direct comparison of hPSC-PC and mouse PC gene expression and a robust method for generating differentiated hPSC-PCs with human-specific gene expression for modeling developmental and degenerative cerebellar disorders.

The Importance of Extracellular Potassium for Differentiation of Cerebellar Purkinje Cells in Tissue Cultures

Despite the fact that cultures of cerebellar granule cells constitute a popular experimental model, the key functional element of the cerebellum, i.e., Purkinje cells (PC), are rarely studied in tissue culture. There are currently serious methodological disagreements relating to the composition of the medium promoting survival of PC and the development of morphology similar to that of in vivo neurons. The development of cerebellar PC in primary tissue cultures at different potassium concentrations was assessed using combinations of vital calcium imaging and immunohistochemical tissue staining for calbindin-28K as a marker for PC. Studies of rat cerebellum were carried out on culture days 7, 14, and 21. Increases in potassium levels in the medium were found to stimulate PC survival, which promoted increases in body size and the development of the dendritic tree of PC. PC demonstrated the intrinsic spontaneous oscillations in free calcium typical for adult PC.

Cend1, a Story with Many Tales: From Regulation of Cell Cycle Progression/Exit of Neural Stem Cells to Brain Structure and Function.

Neural stem/precursor cells (NPCs) generate the large variety of neuronal phenotypes comprising the adult brain. The high diversity and complexity of this organ have its origin in embryonic life, during which NPCs undergo symmetric and asymmetric divisions and then exit the cell cycle and differentiate to acquire neuronal identities. During these processes, coordinated regulation of cell cycle progression/exit and differentiation is essential for generation of the appropriate number of neurons and formation of the correct structural and functional neuronal circuits in the adult brain. Cend1 is a neuronal lineage-specific modulator involved in synchronization of cell cycle exit and differentiation of neuronal precursors. It is expressed all along the neuronal lineage, from neural stem/progenitor cells to mature neurons, and is associated with the dynamics of neuron-generating divisions. Functional studies showed that Cend1 has a critical role during neurogenesis in promoting cell cycle exit and neuronal differentiation. Mechanistically, Cend1 acts via the p53-dependent/Cyclin D1/pRb signaling pathway as well as via a p53-independent route involving a tripartite interaction with RanBPM and Dyrk1B. Upon Cend1 function, Notch1 signaling is suppressed and proneural genes such as Mash1 and Neurogenins 1/2 are induced. Due to its neurogenic activity, Cend1 is a promising candidate therapeutic gene for brain repair, while the Cend1 minimal promoter is a valuable tool for neuron-specific gene delivery in the CNS. Mice with Cend1 genetic ablation display increased NPC proliferation, decreased migration, and higher levels of apoptosis during development. As a result, they show in the adult brain deficits in a range of motor and nonmotor behaviors arising from irregularities in cerebellar cortex lamination and impaired Purkinje cell differentiation as well as a paucity in GABAergic interneurons of the cerebral cortex, hippocampus, and amygdala. Taken together, these studies highlight the necessity for Cend1 expression in the formation of a structurally and functionally normal brain.

Increased Anxiety-Related Behavior, Impaired Cognitive Function and Cellular Alterations in the Brain of Cend1-deficient Mice.

Cend1 is a neuronal-lineage specific modulator involved in coordination of cell cycle exit and differentiation of neuronal precursors. We have previously shown that Cend1−/− mice show altered cerebellar layering caused by increased proliferation of granule cell precursors, delayed radial granule cell migration and compromised Purkinje cell differentiation, leading to ataxic gait and deficits in motor coordination. To further characterize the effects of Cend1 genetic ablation we determined herein a range of behaviors, including anxiety and exploratory behavior in the elevated plus maze (EPM), associative learning in fear conditioning, and spatial learning and memory in the Morris water maze (MWM). We observed significant deficits in all tests, suggesting structural and/or functional alterations in brain regions such as the cortex, amygdala and the hippocampus. In agreement with these findings, immunohistochemistry revealed reduced numbers of γ amino butyric acid (GABA) GABAergic interneurons, but not of glutamatergic projection neurons, in the adult cerebral cortex. Reduced GABAergic interneurons were also observed in the amygdala, most notably in the basolateral nucleus. The paucity in GABAergic interneurons in adult Cend1−/− mice correlated with increased proliferation and apoptosis as well as reduced migration of neuronal progenitors from the embryonic medial ganglionic eminence (MGE), the origin of these cells. Further we noted reduced GABAergic neurons and aberrant neurogenesis in the adult dentate gyrus (DG) of the hippocampus, which has been previously shown to confer spatial learning and memory deficits. Our data highlight the necessity of Cend1 expression in the formation of a structurally and functionally normal brain phenotype.

Expression of the GluA2 subunit of glutamate receptors is required for the normal dendritic differentiation of cerebellar Purkinje cells.

Cerebellar Purkinje cells differentiate the most elaborate dendritic trees among neurons in the brain and constitute the principal part of cerebellar neuronal circuitry. In the present study, we examined the role of the GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors in the dendritic differentiation of Purkinje cells. Since mature Purkinje cells express the GluA2 subunit, AMPA receptors on them exhibit a low Ca2+ permeability. Does this expression of GluA2, leading to the loss of Ca2+ permeability of AMPA receptors, have a positive significance in the dendritic differentiation of Purkinje cells? To answer this question, we introduced GluA2 siRNA into immature Purkinje cells in cerebellar cell cultures using a single-cell electroporation technique. The dendritic elongation and branching, as well as spine formation, were inhibited by GluA2 knockdown in Purkinje cells. GluA2 knockdown augmented the elevation of intracellular Ca2+ concentrations and a higher incidence of oscillation of intracellular Ca2+ concentrations in response to glutamate. These findings suggest that excessive elevation of intracellular Ca2+ concentrations has a negative effect on the dendritic differentiation of Purkinje cells and that the expression of GluA2 inhibits this negative effect in the development of Purkinje cells.

Zfp423/ZNF423 regulates cell cycle progression, the mode of cell division and the DNA-damage response in Purkinje neuron progenitors.

The Zfp423/ZNF423 gene encodes a 30-zinc-finger transcription factor involved in key developmental pathways. Although null Zfp423 mutants develop cerebellar malformations, the underlying mechanism remains unknown. ZNF423 mutations are associated with Joubert Syndrome, a ciliopathy causing cerebellar vermis hypoplasia and ataxia. ZNF423 participates in the DNA-damage response (DDR), raising questions regarding its role as a regulator of neural progenitor cell cycle progression in cerebellar development. To characterize in vivo the function of ZFP423 in neurogenesis, we analyzed allelic murine mutants in which distinct functional domains are deleted. One deletion impairs mitotic spindle orientation, leading to premature cell cycle exit and Purkinje cell (PC) progenitor pool deletion. The other deletion impairs PC differentiation. In both mutants, cell cycle progression is remarkably delayed and DDR markers are upregulated in cerebellar ventricular zone progenitors. Our in vivo evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time strengthens the emerging notion that an impaired DDR may be a key factor in the pathogenesis of JS and other ciliopathies.

MCT8 deficiency in Purkinje cells disrupts embryonic chicken cerebellar development.

Inactivating mutations in the human SLC16A2 gene encoding the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) result in the Allan-Herndon-Dudley syndrome accompanied by severe locomotor deficits. The underlying mechanisms of the associated cerebellar maldevelopment were studied using the chicken as a model. Electroporation of an MCT8-RNAi vector into the cerebellar anlage of a 3-day-old embryo allowed knockdown of MCT8 in Purkinje cell precursors. This resulted in the downregulation of the thyroid hormone-responsive gene RORα and the Purkinje cell-specific differentiation marker LHX1/5 at day 6. MCT8 knockdown also results in a smaller and less complex dendritic tree at day 18 suggesting a pivotal role of MCT8 for cell-autonomous Purkinje cell maturation. Early administration of the thyroid hormone analogue 3,5,3'-triiodothyroacetic acid partially rescued early Purkinje cell differentiation. MCT8-deficient Purkinje cells also induced non-autonomous effects as they led to a reduced granule cell precursor proliferation, a thinner external germinal layer and a loss of PAX6 expression. By contrast, at day 18, the external germinal layer thickness was increased, with an increase in presence of Axonin-1-positive post-mitotic granule cells in the initial stage of radial migration. The concomitant accumulation of presumptive migrating granule cells in the molecular layer, suggests that inward radial migration to the internal granular layer is stalled. In conclusion, early MCT8 deficiency in Purkinje cells results in both cell-autonomous and non-autonomous effects on cerebellar development and indicates that MCT8 expression is essential from very early stages of development, providing a novel insight into the ontogenesis of the Allan-Herndon-Dudley syndrome.

DNA methylation and cerebellar development, the regulation of Notch and Shh pathway

Our purpose is to elucidate the roles of some molecules and DNA methylation in cerebellar development. Immunocytochemistry was performed to investigate the expression of activated Notch1 and Sonic Hedgehog (Shh), as well as DNA methylation during cerebellar development at various ages. Notch1 could be expressed in neural stem cells, newborn neurons and Purkinje cells during cerebellar development. However, Shh could be expressed mainly in developing radial glia or Bergmann cells specifically. DNA methylation was present during cerebellar development, mainly in neural progenitor cells and developing granule cells. The formation and maturation of both Purkinje cells and Bergmann cells are required for the differentiation and migration of granule cells which were bound to DNA methylation. DNA methylation and Shh and Notch signaling pathways function cooperatively during cerebellar development. The Notch pathway mainly contributes to the differentiation of Purkinje cells, but Shh is often involved in the development of Bergmann cells. DNA methylation is necessary for the differentiation and migration of granule cells. The expression of Notch1 and Shh in Purkinje cells and Bergmann cells, following the initiation of DNA methylation in granule cells, indicates that DNA methylation is regulated by Notch and Shh pathways during cerebellar development.

Mutations of the thyroid hormone transporter MCT8 cause prenatal brain damage and persistent hypomyelination.

Mutations in the MCT8 (SLC16A2) gene, encoding a specific thyroid hormone transporter, cause an X-linked disease with profound psychomotor retardation, neurological impairment, and abnormal serum thyroid hormone levels. The nature of the central nervous system damage is unknown. The objective of the study was to define the neuropathology of the syndrome by analyzing brain tissue sections from MCT8-deficient subjects. We analyzed brain sections from a 30th gestational week male fetus and an 11-year-old boy and as controls, brain tissue from a 30th and 28th gestational week male and female fetuses, respectively, and a 10-year-old girl and a 12-year-old boy. Staining with hematoxylin-eosin and immunostaining for myelin basic protein, 70-kDa neurofilament, parvalbumin, calbindin-D28k, and synaptophysin were performed. Thyroid hormone determinations and quantitative PCR for deiodinases were also performed. The MCT8-deficient fetus showed a delay in cortical and cerebellar development and myelination, loss of parvalbumin expression, abnormal calbindin-D28k content, impaired axonal maturation, and diminished biochemical differentiation of Purkinje cells. The 11-year-old boy showed altered cerebellar structure, deficient myelination, deficient synaptophysin and parvalbumin expression, and abnormal calbindin-D28k expression. The MCT8-deficient fetal cerebral cortex showed 50% reduction of thyroid hormones and increased type 2 deiodinase and decreased type 3 deiodinase mRNAs. The following conclusions were reached: 1) brain damage in MCT8 deficiency is diffuse, without evidence of focal lesions, and present from fetal stages despite apparent normality at birth; 2) deficient hypomyelination persists up to 11 years of age; and 3) the findings are compatible with the deficient action of thyroid hormones in the developing brain caused by impaired transport to the target neural cells.

The c-Ski family member and transcriptional regulator Corl2/Skor2 promotes early differentiation of cerebellar Purkinje cells

Purkinje cells (PCs) provide the primary output from the cerebellar cortex, which controls movement and posture, and loss of PCs causes severe cerebellar dysfunction. The mechanisms underlying cell fate determination and early differentiation of PC remain largely unknown. Here we show that the c-Ski family member and transcriptional regulator Corl2 is required for correct differentiation of PCs. In Corl2 knock-out embryos, initial PC specification appeared largely normal, but in a subset of presumptive PCs generated near the ventral border of the PC domain, cell fate choice was compromised and cells showed a mixed identity expressing the interneuron marker Pax2 as well. Additionally, selection and maintenance of the transmitter phenotype was compromised in most developing PCs in the mutants. During later differentiation steps, induction of PC marker genes was significantly suppressed, suggesting that maturation was delayed in the absence of Corl2. Consistently, defects in migration, cell polarization and dendrite formation were observed in mutant PCs, although their axonal trajectories appeared normal. These phenotypes closely resembled those of mutants for Rora, an essential regulator of PC differentiation. However, Rora expression was not significantly changed in the Corl2 mutants, indicating that Corl2 does not simply act upstream of Rora to promote PC differentiation. ChIP experiments revealed that Corl2 bound to the promoter regions of several PC-selective genes, which are also known to be direct downstream targets of RORα. Altogether, our results identified a novel regulatory program of PC differentiation involving Corl2, which might cooperate with the RORα pathway.

Purkinje cells and Bergmann glia are primary targets of the TRα1 thyroid hormone receptor during mouse cerebellum postnatal development

Thyroid hormone is necessary for normal development of the central nervous system, as shown by the severe mental retardation syndrome affecting hypothyroid patients with low levels of active thyroid hormone. The postnatal defects observed in hypothyroid mouse cerebellum are recapitulated in mice heterozygous for a dominant-negative mutation of Thra, the gene encoding the ubiquitous TRα1 receptor. Using CRE/loxP-mediated conditional expression approach, we found that this mutation primarily alters the differentiation of Purkinje cells and Bergmann glia, two cerebellum-specific cell types. These primary defects indirectly affect cerebellum development in a global manner. Notably, the inward migration and terminal differentiation of granule cell precursors is impaired. Therefore, despite the broad distribution of its receptors, thyroid hormone targets few cell types that exert a predominant role in the network of cellular interactions that govern normal cerebellum maturation.

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 Ca(2+) 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.