The relationship between birth timing, circuit wiring, and physiological response properties of cerebellar granule cells.

Cerebellar granule cells (GrCs) are usually regarded as a uniform cell type that collectively expands the coding space of the cerebellum by integrating diverse combinations of mossy fiber inputs. Accordingly, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region have not been reported. The only known cellular property that distinguishes otherwise homogeneous GrCs is the correspondence between GrC birth timing and the depth of the molecular layer to which their axons project. To determine the role birth timing plays in GrC wiring and function, we developed genetic strategies to access early- and late-born GrCs. We initiated retrograde monosynaptic rabies virus tracing from control (birth timing unrestricted), early-born, and late-born GrCs, revealing the different patterns of mossy fiber input to GrCs in vermis lobule 6 and simplex, as well as to early- and late-born GrCs of vermis lobule 6: sensory and motor nuclei provide more input to early-born GrCs, while basal pontine and cerebellar nuclei provide more input to late-born GrCs. In vivo multidepth two-photon Ca2+ imaging of axons of early- and late-born GrCs revealed representations of diverse task variables and stimuli by both populations, with modest differences in the proportions encoding movement, reward anticipation, and reward consumption. Our results suggest neither organized parallel processing nor completely random organization of mossy fiber→GrC circuitry but instead a moderate influence of birth timing on GrC wiring and encoding. Our imaging data also provide evidence that GrCs can represent generalized responses to aversive stimuli, in addition to recently described reward representations.

Oligodendrogenesis and myelination regulate cortical development, plasticity and circuit function.

During cortical development and throughout adulthood, oligodendrocytes add myelin internodes to glutamatergic projection neurons and GABAergic inhibitory neurons. In addition to directing node of Ranvier formation, to enable saltatory conduction and influence action potential transit time, oligodendrocytes support axon health by communicating with axons via the periaxonal space and providing metabolic support that is particularly critical for healthy ageing. In this review we outline the timing of oligodendrogenesis in the developing mouse and human cortex and describe the important role that oligodendrocytes play in sustaining and modulating neuronal function. We also provide insight into the known and speculative impact that myelination has on cortical axons and their associated circuits during the developmental critical periods and throughout life, particularly highlighting their life-long role in learning and remembering.

A TRPC5-regulated calcium signaling pathway controls dendrite patterning in the mammalian brain.

Transient receptor potential (TRP) channels have been implicated as sensors of diverse stimuli in mature neurons. However, developmental roles for TRP channels in the establishment of neuronal connectivity remain largely unexplored. Here, we identify an essential function for TRPC5, a member of the canonical TRP subfamily, in the regulation of dendrite patterning in the mammalian brain. Strikingly, TRPC5 knockout mice harbor long, highly branched granule neuron dendrites with impaired dendritic claw differentiation in the cerebellar cortex. In vivo RNAi analyses suggest that TRPC5 regulates dendrite morphogenesis in the cerebellar cortex in a cell-autonomous manner. Correlating with impaired dendrite patterning in the cerebellar cortex, behavioral analyses reveal that TRPC5 knockout mice have deficits in gait and motor coordination. Finally, we uncover the molecular basis of TRPC5's function in dendrite patterning. We identify the major protein kinase calcium/calmodulin-dependent kinase II ß (CaMKIIß) as a critical effector of TRPC5 function in neurons. Remarkably, TRPC5 forms a complex specifically with CaMKIIß, but not the closely related kinase CaMKIIα, and thereby induces the CaMKIIß-dependent phosphorylation of the ubiquitin ligase Cdc20-APC at the centrosome. Accordingly, centrosomal CaMKIIß signaling mediates the ability of TRPC5 to regulate dendrite morphogenesis in neurons. Our findings define a novel function for TRPC5 that couples calcium signaling to a ubiquitin ligase pathway at the centrosome and thereby orchestrates dendrite patterning and connectivity in the brain.

Lkb1 regulates granule cell migration and cortical folding of the cerebellar cortex.

Cerebellar growth and foliation require the Hedgehog-driven proliferation of granule cell precursors (GCPs) in the external granule layer (EGL). However, that increased or extended GCP proliferation generally does not elicit ectopic folds suggests that additional determinants control cortical expansion and foliation during cerebellar development. Here, we find that genetic loss of the serine-threonine kinase Liver Kinase B1 (Lkb1) in GCPs increased cerebellar cortical size and foliation independent of changes in proliferation or Hedgehog signaling. This finding is unexpected given that Lkb1 has previously shown to be critical for Hedgehog pathway activation in cultured cells. Consistent with unchanged proliferation rate of GCPs, the cortical expansion of Lkb1 mutants is accompanied by thinning of the EGL. The plane of cell division, which has been implicated in diverse processes from epithelial surface expansions to gyrification of the human cortex, remains unchanged in the mutants when compared to wild-type controls. However, we find that Lkb1 mutants display delayed radial migration of post-mitotic GCPs that coincides with increased cortical size, suggesting that aberrant cell migration may contribute to the cortical expansion and increase foliation. Taken together, our results reveal an important role for Lkb1 in regulating cerebellar cortical size and foliation in a Hedgehog-independent manner.

Migration of Interneuron Precursors in the Nascent Cerebellar Cortex.

The cerebellum arguably constitutes one of the best characterized central nervous circuits, and its structure, cellular function, and histogenesis have been described in exceptional quantitative detail. A notable exception to this is the development of its inhibitory interneurons, and in particular the extensive migrations of future basket and stellate cells. Here, we used acute slices from 8-day-old mice to assess the migration of Pax2-EGFP-tagged precursors of these cells en route to the molecular layer during their transit through the nascent cerebellar cortex. We document that movement of these cells is highly directed. Their speed and directional persistence are larger in the nascent granule cell layer than in the molecular layer. And they migrate periodically, with periods of effective, directed translocation separated by bouts of rather local movement. Finally, we document that the arrangement of these cells in the adult molecular layer is characterized by clustering. These data are discussed with a focus on potential generative mechanisms for the developmental pattern observed.

Development and Evolution of Cerebral and Cerebellar Cortex.

Cerebral cortex and cerebellar cortex both vary enormously across species in their size and complexity of convolutions. We discuss the development and evolution of cortical structures in terms of anatomy and functional organization. We propose that the distinctive shapes of cerebral and cerebellar cortex can be explained by relatively few developmental processes, notably including mechanical tension along axons and dendrites. Regarding functional organization, we show how maps of myelin content in cerebral cortex are evolutionarily conserved across primates but differ in the proportion of cortex devoted to sensory, cognitive, and other functions. We summarize recent progress and challenges in (i) parcellating cerebral cortex into a mosaic of distinct areas, (ii) distinguishing cortical areas that correspond across species from those that are present in one species but not another, and (iii) using this information along with surface-based interspecies registration to gain deeper insights into cortical evolution. We also comment on the methodological challenges imposed by the differences in anatomical and functional organization of cerebellar cortex relative to cerebral cortex.

Analysis of hedgehog signaling in cerebellar granule cell precursors in a conditional Nsdhl allele demonstrates an essential role for cholesterol in postnatal CNS development.

NSDHL is a 3ß-hydroxysterol dehydrogenase that is involved in the removal of two C-4 methyl groups in one of the later steps of cholesterol biosynthesis. Mutations in the gene encoding the enzyme are responsible for the X-linked, male lethal mouse mutations bare patches and striated, as well as most cases of human CHILD syndrome. Rare, hypomorphic NSDHL mutations are also associated with X-linked intellectual disability in males with CK syndrome. Since hemizygous male mice with Nsdhl mutations die by midgestation, we generated a conditional targeted Nsdhl mutation (Nsdhl(tm1.1Hrm)) to investigate the essential role of cholesterol in the early postnatal CNS. Ablation of Nsdhl in radial glia using GFAP-cre resulted in live-born, normal appearing affected male pups. However, the pups develop overt ataxia by postnatal day 8-10 and die shortly thereafter. Histological abnormalities include progressive loss of cortical and hippocampal neurons, as well as deficits in the proliferation and migration of cerebellar granule precursors and subsequent massive apoptosis of the cerebellar cortex. We replicated the granule cell precursor proliferation defect in vitro and demonstrate that it results from defective signaling by SHH. Furthermore, this defect is almost completely rescued by supplementation of the culture media with exogenous cholesterol, while methylsterol accumulation above the enzymatic block appears to be associated with increased cell death. These data support the absolute requirement for cholesterol synthesis in situ once the blood-brain-barrier forms and cholesterol transport to the fetus is abolished. They further emphasize the complex ramifications of cholesterogenic enzyme deficiency on cellular metabolism.

Functional regionalization of the teleost cerebellum analyzed in vivo.

There has been accumulating evidence for a regionalized organization of the cerebellum, which was mostly deduced from anatomical mapping of axonal projections of cerebellar afferents. A likewise regionalization of the cerebellar output has been suggested from lesion studies and dye-tracer experiments, but its physiological targets as well as the functional relevance of such an output regionalization are less clear. Ideally, such functional regionalization should be proven noninvasively in vivo. We here provide evidence for such a regionalization of the output from the cerebellar cortex by genetically encoded transneuronal mapping of efferent circuits of zebrafish Purkinje neurons. These identified circuits correspond to distinct regionalized Purkinje cell activity patterns in freely behaving zebrafish larvae during the performance of cerebellar-dependent behaviors. Furthermore, optogenetic interrogation of selected Purkinje cell regions during animal behavior confirms the functional regionalization of Purkinje cell efferents and reveals their contribution to behavior control as well as their function in controlling lateralized behavioral output. Our findings reveal how brain compartments serve to fulfill a multitude of functions by dedicating specialized efferent circuits to distinct behavioral tasks.

Maturation, Refinement, and Serotonergic Modulation of Cerebellar Cortical Circuits in Normal Development and in Murine Models of Autism.

The formation of the complex cerebellar cortical circuits follows different phases, with initial synaptogenesis and subsequent processes of refinement guided by a variety of mechanisms. The regularity of the cellular and synaptic organization of the cerebellar cortex allowed detailed studies of the structural plasticity mechanisms underlying the formation of new synapses and retraction of redundant ones. For the attainment of the monoinnervation of the Purkinje cell by a single climbing fiber, several signals are involved, including electrical activity, contact signals, homosynaptic and heterosynaptic interaction, calcium transients, postsynaptic receptors, and transduction pathways. An important role in this developmental program is played by serotonergic projections that, acting on temporally and spatially regulated postsynaptic receptors, induce and modulate the phases of synaptic formation and maturation. In the adult cerebellar cortex, many developmental mechanisms persist but play different roles, such as supporting synaptic plasticity during learning and formation of cerebellar memory traces. A dysfunction at any stage of this process can lead to disorders of cerebellar origin, which include autism spectrum disorders but are not limited to motor deficits. Recent evidence in animal models links impairment of Purkinje cell function with autism-like symptoms including sociability deficits, stereotyped movements, and interspecific communication by vocalization.

Autophagy-related gene Atg5 is essential for astrocyte differentiation in the developing mouse cortex.

Astrocyte differentiation is essential for late embryonic brain development, and autophagy is active during this process. However, it is unknown whether and how autophagy regulates astrocyte differentiation. Here, we show that Atg5, which is necessary for autophagosome formation, regulates astrocyte differentiation. Atg5 deficiency represses the generation of astrocytes in vitro and in vivo. Conversely, Atg5 overexpression increases the number of astrocytes substantially. We show that Atg5 activates the JAK2-STAT3 pathway by degrading the inhibitory protein SOCS2. The astrocyte differentiation defect caused by Atg5 loss can be rescued by human Atg5 overexpression, STAT3 overexpression, and SOCS2 knockdown. Together, these data demonstrate that Atg5 regulates astrocyte differentiation, with potential implications for brain disorders with autophagy deficiency.

Tri-layer wrinkling as a mechanism for anchoring center initiation in the developing cerebellum.

During cerebellar development, anchoring centers form at the base of each fissure and remain fixed in place while the rest of the cerebellum grows outward. Cerebellar foliation has been extensively studied; yet, the mechanisms that control anchoring center initiation and position remain insufficiently understood. Here we show that a tri-layer model can predict surface wrinkling as a potential mechanism to explain anchoring center initiation and position. Motivated by the cerebellar microstructure, we model the developing cerebellum as a tri-layer system with an external molecular layer and an internal granular layer of similar stiffness and a significantly softer intermediate Purkinje cell layer. Including a weak intermediate layer proves key to predicting surface morphogenesis, even at low stiffness contrasts between the top and bottom layers. The proposed tri-layer model provides insight into the hierarchical formation of anchoring centers and establishes an essential missing link between gene expression and evolution of shape.

Stereological study of the effects of maternal diabetes on cerebellar cortex development in rat.

Diabetes during pregnancy is associated with the deficits in balance and motor coordination and altered social behaviors in offspring. In the present study, we have investigated the effect of maternal diabetes and insulin treatment on the cerebellar volume and morphogenesis of the cerebellar cortex of rat neonates during the first two postnatal weeks. Sprague Dawley female rats were maintained diabetic from a week before pregnancy through parturition. At the end of pregnancy, the male offspring euthanized on postnatal days (P) 0, 7, and 14. Cavalieri's principle and fractionator methods were used to estimate the cerebellar volume, the thickness and the number of cells in the different layers of the cerebellar cortex. In spite of P0, there was a significant reduction in the cerebellar volume and the thickness of the external granule, molecular, and internal granule layers between the diabetic and the control animals. In diabetic group, the granular and purkinje cell densities were increased at P0. Moreover, the number of granular and purkinje cells in the cerebellum of diabetic neonates was reduced in comparison with the control group at P7 and P14. There were no significant differences in either the volume and thickness or the number of cells in the different layers of the cerebellar cortex between the insulin-treated diabetic group and controls. Our data indicate that diabetes in pregnancy disrupts the morphogenesis of cerebellar cortex. This dysmorphogenesis may be part of the cascade of events through which diabetes during pregnancy affects motor coordination and social behaviors in offspring.

Altered expression and localization of synaptophysin in developing cerebellar cortex of neonatal rats due to maternal diabetes mellitus.

There is sufficient evidence that diabetes during pregnancy is associated with a higher risk of neurodevelopmental anomalies including learning deficits, behavioral problems and motor dysfunctions in the offspring. Synaptophysin (SYP) is an integral membrane protein of synaptic vesicles and is considered as a marker for synaptogenesis and synaptic density. This study aimed to examine the effects of maternal diabetes in pregnancy on the expression and localization of SYP in the developing rat cerebellum. Wistar female rats were maintained diabetic from a week before pregnancy through parturition and male offspring was euthanized at postnatal day (P) 0, 7, and 14. The results revealed a significant down-regulation in the mRNA expression of SYP in the offspring born to diabetic animals at both P7 and P14 (P < 0.05 each). One week after birth, there was a significant reduction in the localization of SYP expression in the external granular (EGL) and in the molecular (ML) layers of neonates born to diabetic animals (P < 0.05 each). We also found a marked decrease in the expression of SYP in all of the cerebellar cortical layers of STZ-D group pups at P14 (P < 0.05 each). Moreover, our results revealed no significant changes in either expression or localization of SYP in insulin-treated group pups when compared with the controls (P ≥ 0.05 each). The present study demonstrated that maternal diabetes has adverse effects on the synaptogenesis in the offspring's cerebellum. Furthermore, the rigid maternal blood glucose control in the most cases normalized these negative impacts.

Tissue plasminogen activator regulates Purkinje neuron development and survival.

The cerebellar cortex is centrally involved in motor coordination and learning, and its sole output is provided by Purkinje neurons (PNs). Growth of PN dendrites and their major synaptic input from granule cell parallel fiber axons takes place almost entirely in the first several postnatal weeks. PNs are more vulnerable to cell death than most other neurons, but the mechanisms remain unclear. We find that the homozygous nervous (nr) mutant mouse's 10-fold-increased cerebellar tissue plasminogen activator (tPA), a part of the tPA/plasmin proteolytic system, influences several different molecular mechanisms, each regulating a key aspect of postnatal PN development, followed by selective PN necrosis, as follows. (i) Excess endogenous or exogenous tPA inhibits dendritic growth in vivo and in vitro by activating protein kinase Cγ and phosphorylation of microtubule-associated protein 2. (ii) tPA/plasmin proteolysis impairs parallel fiber-PN synaptogenesis by blocking brain-derived neurotrophic factor/tyrosine kinase receptor B signaling. (iii) Voltage-dependent anion channel 1 (a mitochondrial and plasma membrane protein) bound with kringle 5 (a peptide derived from the excess plasminogen) promotes pathological enlargement and rounding of PN mitochondria, reduces mitochondrial membrane potential, and damages plasma membranes. These abnormalities culminate in young nr PN necrosis that can be mimicked in wild-type PNs by exogenous tPA injection into cerebellum or prevented by endogenous tPA deletion in nr:tPA-knockout double mutants. In sum, excess tPA/plasmin, through separate downstream molecular mechanisms, regulates postnatal PN dendritogenesis, synaptogenesis, mitochondrial structure and function, and selective PN viability.

Cortical-layer-specific effects of PACAP and tPA on interneuron migration during post-natal development of the cerebellum.

During early post-natal development of the cerebellum, granule neurons (GN) execute a centripetal migration toward the internal granular layer, whereas basket and stellate cells (B/SC) migrate centrifugally to reach their final position in the molecular layer (ML). We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates in vitro the expression and release of the serine protease tissue-type plasminogen activator (tPA) from GN, but the coordinated role of PACAP and tPA during interneuron migration has not yet been investigated. Here, we show that endogenous PACAP is responsible for the transient arrest phase of GN at the level of the Purkinje cell layer (PCL) but has no effect on B/SC. tPA is devoid of direct effect on GN motility in vitro, although it is widely distributed along interneuron migratory routes in the ML, PCL, and internal granular layer. Interestingly, plasminogen activator inhibitor 1 reduces the migration speed of GN in the ML and PCL, and that of B/SC in the ML. Taken together, these results reveal for the first time that tPA facilitates the migration of both GN and fast B/SC at the level of their intersection in the ML through degradation of the extracellular matrix. Crucial role of tissue plasminogen activator (tPA) in interneuron migration. Interneuron migration is a critical step for normal establishment of neuronal network. This study indicates that, in the post-natal cerebellum, tPA facilitates the opposite migration of immature excitatory granule neurons (GN) and immature inhibitory basket/stellate cells (B/SC) along the same migratory route. These data show that tPA exerts a pivotal role in neurodevelopment.

The similar expression pattern of MHC class I molecules in human and mouse cerebellar cortex.

The major histocompatibility complex (MHC) class I molecules are considered to be important in the immune system. However, the results reported in the past decade indicate that they also play important roles in the central nervous system. Here we examined the expression of MHC I and ß2-microglobulin (ß2m) in human and mouse cerebellar cortex. The results show that MHC I molecules are expressed both in human and mouse cerebellar cortex during brain development. The expression of H-2K(b)/D(b) is gradually increased with the development of mouse cerebellar cortex, but finally decreased to a very low level. Similarly, the expression of HLA-B/C genes is increased in developing human cerebellar cortex, but decreased after birth. The spatial and temporal expression of ß2m overlaps mostly with that of HLA-B/C molecules, and they are co-expressed in Purkinje cells. Our findings provide a fundamental basis to reveal the functions of neuronal MHC class I molecules in the development of human cerebellum.

Differential regulation of GLT-1/EAAT2 gene expression by NF-κB and N-myc in male mouse brain during postnatal development.

The synaptic glutamate level homeostasis is mainly maintained by the astrocytes membrane bound glutamate transporter type-1 (GLT-1/EAAT2). Alterations in its expression during development and aging and the underlying mechanisms are not well studied. Here, we report that NF-κB interaction was highest in both cerebral and cerebellar cortices at day 15 when compared with that at day 0 during development, and it further declined significantly in day 45, and remained unchanged in 20 and 70 weeks mice. On the other hand, N-myc interaction was highest at 0 day which significantly declined at 15-day and interestingly remained unaltered at later ages in both the cortices. This age dependent reciprocal pattern of NF-κB and N-myc interactions with their cognate GLT-1 promoter sequences was further correlated with GLT-1 protein and transcript levels. We found that higher NF-κB interaction with its cognate GLT-1 promoter sequences correlates with up-regulation whereas the higher N-myc interaction correlates with down-regulation of GLT-1 expression during postnatal developmental age up to 15 day, however, such phenomenon was not found in the higher ages from day 45 to 70 weeks. Thus our data suggests a postnatal development- and age dependent differential interaction of transcription factors NF-κB and N-myc to their respective sequences and they act as positive and negative regulator, respectively of GLT-1 gene expression in the brain during early developmental period in both cerebral and cerebellar cortices which might be different in aging of mice.

Inducible and reversible regulation of endogenous gene in mouse.

Methods for generating loss-of-function mutations, such as conventional or conditional gene knockout, are widely used in deciphering gene function in vivo. By contrast, inducible and reversible regulation of endogenous gene expression has not been well established. Using a mouse model, we demonstrate that a chimeric transcriptional repressor molecule (tTS) can reversibly inhibit the expression of an endogenous gene, Nmyc. In this system, a tetracycline response element (TRE) artificially inserted near the target gene's promoter region turns the gene on and off in a tetracycline-inducible manner. Nmyc(TRE) mice were generated by inserting a TRE into the first intron of Nmyc by the knockin technique. Nmyc(TRE) mice were crossed to tTS transgenic mice to produce Nmyc(TRE/TRE): tTS embryos. In these embryos, tTS blocked Nmyc expression, and embryonic lethality was observed at E11.5d. When the dam was exposed to drinking water containing doxycycline (dox), normal endogenous Nmyc expression was rescued, and the embryo survived to birth. This novel genetic modification strategy based on the tTS-dox system for inducible and reversible regulation of endogenous mouse genes will be a powerful tool to investigate target genes that cause embryonic lethality or other defects where reversible regulation or temporary shutdown of the target gene is needed.

Impact of varying maternal dietary folate intake on cerebellar cortex histomorphology and cell density in offspring rats.

The cerebellum has a long, protracted developmental period that spans from the embryonic to postnatal periods; as a result, it is more sensitive to intrauterine and postnatal insults like nutritional deficiencies. Folate is crucial for foetal and early postnatal brain development; however, its effects on cerebellar growth and development are unknown. The aim of this study was to examine the effects of maternal folate intake on the histomorphology and cell density of the developing cerebellum. Twelve adult female rats (rattus norvegicus) were randomly assigned to one of four premixed diet groups: standard (2 mg/kg), folate-deficient (0 mg/kg), folate-supplemented (8 mg/kg) or folate supra-supplemented (40 mg/kg). The rats started their diets 14 days before mating and consumed them throughout pregnancy and lactation. On postnatal days 1, 7, 21 and 35, five pups from each group were sacrificed, and their brains were processed for light microscopic analysis. Histomorphology and cell density of the external granule, molecular, Purkinje and internal granule layers were obtained. The folate-deficient diet group had smaller, dysmorphic cells and significantly lower densities of external granule, molecular, Purkinje and internal granule cells. Although the folate-enriched groups had greater cell densities than the controls, the folate-supplemented group had considerably higher cell densities than the supra-supplemented group. The folate supra-supplemented group had ectopic Purkinje cells in the internal granule cell layer. These findings imply that a folate-deficient diet impairs cellular growth and reduces cell density in the cerebellar cortex. On the other hand, folate supplementation increases cell densities, but there appears to be an optimal dose of supplementation since excessive folate levels may be detrimental.

Clustered fine compartmentalization of the mouse embryonic cerebellar cortex and its rearrangement into the postnatal striped configuration.

Compartmentalization is essential for a brain area to be involved in different functions through topographic afferent and efferent connections that reflect this organization. The adult cerebellar cortex is compartmentalized into longitudinal stripes, in which Purkinje cells (PCs) have compartment-specific molecular expression profiles. How these compartments form during development is generally not understood. To investigate this process, we focused on the late developmental stages of the cerebellar compartmentalization that occur from embryonic day 17.5 (E17.5), when embryonic compartmentalization is evidently observed, to postnatal day 6 (P6), when adult-type compartmentalization begins to be established. The transformation between these compartmentalization patterns was analyzed by mapping expression patterns of several key molecular markers in serial cerebellar sections in the mouse. A complete set of 54 clustered PC subsets, which had different expression profiles of FoxP2, PLCß4, EphA4, Pcdh10, and a reporter molecule of the 1NM13 transgenic mouse strain, were distinguished in three-dimensional space in the E17.5 cerebellum. Following individual PC subsets during development indicated that these subsets were rearranged from a clustered and multilayered configuration to a flattened, single-layered and striped configuration by means of transverse slide, longitudinal split, or transverse twist spatial transformations during development. The Purkinje cell-free spaces that exist between clusters at E17.5 become granule cell raphes that separate striped compartments at P6. The results indicate that the ∼50 PC clusters of the embryonic cerebellum will ultimately become the longitudinal compartments of the adult cerebellum after undergoing various peri- and postnatal transformations that alter their relative spatial relationships.