Early Cerebellar Development Research Articles

Self-supervised learning with application for infant cerebellum segmentation and analysis

Accurate tissue segmentation is critical to characterize early cerebellar development in the first two postnatal years. However, challenges in tissue segmentation arising from tightly-folded cortex, low and dynamic tissue contrast, and large inter-site data heterogeneity have limited our understanding of early cerebellar development. In this paper, we propose an accurate self-supervised learning framework for infant cerebellum segmentation. We validate its accuracy using 358 subjects from three datasets. Our results suggest the first six months exhibit the most rapid and dynamic changes, with gray matter (GM) playing a dominant role in cerebellar growth over white matter (WM). We also find both GM and WM volumes are larger in males than females, and GM and WM volumes are larger in autistic males than neurotypical males. Application of our method to a larger population will fuel more cerebellar studies, ultimately advancing our comprehension of its structure and function in neurotypical and disordered development.

Models of Purkinje cell dendritic tree selection during early cerebellar development.

We investigate the relationship between primary dendrite selection of Purkinje cells and migration of their presynaptic partner granule cells during early cerebellar development. During postnatal development, each Purkinje cell grows more than three dendritic trees, from which a primary tree is selected for development, whereas the others completely retract. Experimental studies suggest that this selection process is coordinated by physical and synaptic interactions with granule cells, which undergo a massive migration at the same time. However, technical limitations hinder continuous experimental observation of multiple cell populations. To explore possible mechanisms underlying this selection process, we constructed a computational model using a new computational framework, NeuroDevSim. The study presents the first computational model that simultaneously simulates Purkinje cell growth and the dynamics of granule cell migrations during the first two postnatal weeks, allowing exploration of the role of physical and synaptic interactions upon dendritic selection. The model suggests that interaction with parallel fibers is important to establish the distinct planar morphology of Purkinje cell dendrites. Specific rules to select which dendritic trees to keep or retract result in larger winner trees with more synaptic contacts than using random selection. A rule based on afferent synaptic activity was less effective than rules based on dendritic size or numbers of synapses.

Issue Information

The FEBS JournalVolume 290, Issue 11 p. 2783-2785 Issue Cover & InformationFree Access Issue Information First published: 07 June 2023 https://doi.org/10.1111/febs.16498AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Cover Illustration Cerebellar reconstruction of neurons with a history of Olig3 expression, visualized by tdTomato fluorescence and light-sheet microscopy (image provided by Luis Rodrigo Hernandez-Miranda and co-authors). See the State-of-the-Art Review on regulation of early cerebellar development (pages 2786–2804). Volume290, Issue11June 2023Pages 2783-2785 RelatedInformation

Models of Purkinje cell dendritic tree selection during early cerebellar development

Models of Purkinje cell dendritic tree selection during early cerebellar development

Models of Purkinje cell dendritic tree selection during early cerebellar development

Models of Purkinje cell dendritic tree selection during early cerebellar development

Potential role of TGFΒ and autophagy in early cerebellum development

During development, the interconnected generation of various neural cell types within the cerebellar primordium is essential. Over embryonic (E) days E9−E13, Purkinje cells (PCs), and cerebellar nuclei (CN) neurons are among the created primordial neurons. The molecular and cellular mechanisms fundamental for the early cerebellar neurogenesis, migration/differentiation, and connectivity are not clear yet. Autophagy has a vital role in controlling cellular phenotypes, such as epithelial-to-mesenchymal transition (EMT) and endothelial to mesenchymal transition (EndMT). Transforming growth factor-beta 1 (TGF-β1) is the main player in pre-and postnatal development and controlling cellular morphological type via various mechanisms, such as autophagy. Thus, we hypothesized that TGF-β1 may regulate early cerebellar development by modifying the levels of cell adhesion molecules (CAMs) and consequently autophagy pathway in the mouse cerebellar primordium. We demonstrated the stimulation of the canonical TGF-β1 signaling pathway at the point that concurs with the generation of the nuclear transitory zone and PC plate in mice. Furthermore, our data show that the stimulated TGF-β1 signaling pathway progressively and chronologically could upregulate the expression of β-catenin (CTNNB1) and N-cadherin (CDH2) with the most expression at E11 and E12, leading to upregulation of chromodomain helicase DNA binding protein 8 (CDH8) and neural cell adhesion molecule 1 (NCAM1) expression, at E12 and E13. Finally, we demonstrated that the stimulated TGF-β signaling pathway may impede the autophagic flux at E11/E12. Nevertheless, basal autophagy flux happens at earlier developmental phases from E9−E10. Our study determined potential role of the TGF-β signaling and its regulatory impacts on autophagic flux during cerebellar development and cadherin expression, which can facilitate the proliferation, migration/differentiation, and placement of PCs and the CN neurons in their designated areas.

Phenotypic, metabolic, and biogenesis properties of human stem cell-derived cerebellar spheroids

Human cerebellum consists of high density and complexity of neurons. Thus, it is challenging to differentiate cerebellar-like organoids with similar cellular markers and function to the human brain. Our previous study showed that the combination of retinoic acid (RA), Wingless/integrated (Wnt) activator, and Sonic Hedgehog (SHH) activator promotes cerebellar differentiation from human induced pluripotent stem cells (hiPSCs). This study examined phenotypic, metabolic, and biogenesis in early cerebellar development. Cerebellum spheroids were differentiated from human iPSK3 cells. During day 7–14, RA and Wnt activator CHIR99021 were used and SHH activator purmorphamine (PMR) was added later to promote ventralization. Gene expression for early cerebellar layer markers, metabolism, and extracellular vesicle (EV) biogenesis were characterized. Zinc-induced neurotoxicity was investigated as a proof-of-concept of neurotoxicity study. Flow cytometry results showed that there was no significant difference in NEPH3, PTF1A, OLIG2, and MATH1 protein expression between RCP (RA-CHIR-PMR) versus the control condition. However, the expression of cerebellar genes for the molecular layer (BHLE22), the granule cell layer (GABRB2, PAX6, TMEM266, KCNIP4), the Bergmann glial cells (QK1, DAO), and the Purkinje cell layer (ARHGEF33, KIT, MX1, MYH10, PPP1R17, SCGN) was significantly higher in the RCP condition than the control. The shift in metabolic pathways toward glycolysis was observed for RCP condition. The EV biogenesis marker expression was retained. Mild zinc-induced neurotoxicity may exist when zinc exposure exceeds 1.0 µM. RCP treatment can promote specific cerebellar-like differentiation from hiPSCs indicated by gene expression of early cerebellar markers and regionally enriched genes. The higher cerebellar marker expression is accompanied by the elevated glycolysis with the retained EV biogenesis. This study should advance the understanding of biomarkers during early cerebellar development for cerebellum organoid engineering and neurotoxicity study.

Regulation of early cerebellar development.

The study of cerebellar development has been at the forefront of neuroscience since the pioneering work of Wilhelm His Sr., Santiago Ramón y Cajal and many others since the 19th century. They laid the foundation to identify the circuitry of the cerebellum, already revealing its stereotypic three-layered cortex and discerning several of its neuronal components. Their work was fundamental in the acceptance of the neuron doctrine, which acknowledges the key role of individual neurons in forming the basic units of the nervous system. Increasing evidence shows that the cerebellum performs a variety of homeostatic and higher order neuronal functions beyond the mere control of motor behaviour. Over the last three decades, many studies have revealed the molecular machinery that regulates distinct aspects of cerebellar development, from the establishment of a cerebellar anlage in the posterior brain to the identification of cerebellar neuron diversity at the single cell level. In this review, we focus on summarizing our current knowledge on early cerebellar development with a particular emphasis on the molecular determinants that secure neuron specification and contribute to the diversity of cerebellar neurons.

Early Cerebellar Development in Relation to the Trigeminal System.

Despite the wealth of knowledge of adult cerebellar connectivity, little is known about the developmental mechanisms that underpin its development. Early connectivity is important because it is the foundation of the neural networks crucial for neuronal function and serves as a scaffold on which later tracts form. Conventionally, it is believed that afferents from the vestibular system are the first to invade the cerebellum, at embryonic days (E) 11-E12/13 in mice, where they target the new born Purkinje cells. However, we have demonstrated that pioneer axons that originate from the trigeminal ganglia are already present in the cerebellar primordium by E9, a stage at which afferents from the vestibular ganglia have not yet reached the brainstem, where they target neurons of the cerebellar nuclei. An early-born subset of cerebellar nuclei may be derived from the mesencephalon. These may be the target of the earliest pioneer axons. They form the early connectivity at the rostral end. This is consistent with the notion that the formation of the antero-posterior axis follows a rostro-caudal sequence. The finding that trigeminal ganglion-derived pioneer axons enter the cerebellar primordium before Purkinje cells are born and target the cerebellar nuclei, reveals a novel perspective on the development of early cerebellar connectivity.

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.

The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish

Joubert syndrome is characterized by unique malformation of the cerebellar vermis. More than thirty Joubert syndrome genes have been identified, including ARL13B. However, its role in cerebellar development remains unexplored. We found that knockdown or knockout of arl13b impaired balance and locomotion in zebrafish larvae. Granule cells were selectively reduced in the corpus cerebelli, a structure homologous to the mammalian vermis. Purkinje cell progenitors were also selectively disturbed dorsomedially. The expression of atoh1 and ptf1, proneural genes of granule and Purkinje cells, respectively, were selectively down-regulated along the dorsal midline of the cerebellum. Moreover, wnt1, which is transiently expressed early in cerebellar development, was selectively reduced. Intriguingly, activating Wnt signaling partially rescued the granule cell defects in arl13b mutants. These findings suggested that Arl13b is necessary for the early development of cerebellar granule and Purkinje cells. The arl13b-deficient zebrafish can serve as a model organism for studying Joubert syndrome.

Quantitative Analysis of the Timing of Development of the Cerebellum and Precerebellar Nuclei in Monotremes, Metatherians, Rodents, and Humans.

We have used a quantitative statistical approach to compare the pace of development in the cerebellum and precerebellar systems relative to body size in monotremes and metatherians with that in eutherians (rodents and humans). Embryos, fetuses, and early postnatal mammals were scored on whether key structural events had been reached in the development of the cerebellum itself (CC-corpus cerebelli; 10 milestones), or the pontine and inferior olivary precerebellar nuclear groups (PC; 4 milestones). We found that many early cerebellar and precerebellar milestones (e.g., formation of Purkinje cell layer and deep cerebellar nuclei) were reached at a smaller absolute body length in both metatherians and eutherians together, compared to monotremes. Some later milestones (e.g., formation of the external granular layer and primary fissuration) were reached at a smaller body length in metatherians than eutherians. When the analysis was performed with proportional body length expressed as a natural log-transformed ratio of length at birth, milestones were reached at a much smaller proportional body length in rodents and humans than in the metatherians or monotremes. The findings are consistent with the slower pace of metabolic activity and embryonic development in monotremes. They also indicate slightly advanced maturation of some early features of the cerebellum in some metatherians (i.e., early cerebellar development in dasyurids relative to body size), but do not support the notion of an accelerated development of the cerebellum to cope with the demands of early birth. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1998-2013, 2020. © 2019 American Association for Anatomy.

Light-at-night exposure affects brain development through pineal allopregnanolone-dependent mechanisms.

The molecular mechanisms by which environmental light conditions affect cerebellar development are incompletely understood. We showed that circadian disruption by light-at-night induced Purkinje cell death through pineal allopregnanolone (ALLO) activity during early life in chicks. Light-at-night caused the loss of diurnal variation of pineal ALLO synthesis during early life and led to cerebellar Purkinje cell death, which was suppressed by a daily injection of ALLO. The loss of diurnal variation of pineal ALLO synthesis induced not only reduction in pituitary adenylate cyclase-activating polypeptide (PACAP), a neuroprotective hormone, but also transcriptional repression of the cerebellar Adcyap1 gene that produces PACAP, with subsequent Purkinje cell death. Taken together, pineal ALLO mediated the effect of light on early cerebellar development in chicks.

Potential deficit from decreased cerebellar granule cell migration in serine racemase-deficient mice is reversed by increased expression of GluN2B and elevated levels of NMDAR agonists.

Inward migration of cerebellar granule cells (CGCs) after birth is critical for lamination in the cerebellar cortex. N-methyl-d-aspartate (NMDA) subtype of glutamate receptor (NMDAR) tethering CGCs into Bergmann glial fibers mediates the inward movement during the glial-dependent migratory phase. Activation of NMDAR depends on simultaneous binding of the GluN2 subunit by glutamate, and of the GluN1 subunit by d-serine or glycine; d-serine is believed to be an endogenous ligand of NMDAR. We hypothesized that lamination of the cerebellar cortex may be compromised in Srr (the gene for serine racemase (SR)) mutated mice (Srrnull) because of significantly low levels of d-serine per se. Indeed, the external germinal cell layer (EGL) in Srrnull was thicker than in sibling wild-type (WT) mice on postnatal day7 (P7), which accords with decreased CGC migration in Srrnull mice. However, the cerebellar laminar structure in Srrnull mice was normal on P12 and later. Feeding d-serine to pregnant mice abrogated the increased EGL thickness in Srrnull mice on P7. To determine the underlying mechanism of abnormal laminar structure during cerebellar development in Srrnull mice, we examined NMDAR subunits and their ligands. We found increased GluN2B on P10 and increased glycine during P7-12 in the cerebellar homogenates from Srrnull mice compared with the corresponding values from sibling WT mice. In summary, the study revealed how the potential defect in early cerebellar development caused by Srr mutation is circumvented by a compensatory mechanism. This knowledge advances understanding of the adaptation of cerebellar development under the condition of Srr mutation.

The Role of MicroRNAs in Cerebellar Development and Autism Spectrum Disorder During Embryogenesis.

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules with wide-ranging and subtle effects on protein production. Their activity during the development of the cerebellum provides a valuable exemplar of how non-coding molecules may assist the development and function of the central nervous system and drive neurodevelopmental disorders. Three distinct aspects of miRNA contribution to early cerebellar development will here be reviewed. Aspects are the establishment of the cerebellar anlage, the generation and maturation of at least two principal cell types of the developing cerebellar microcircuit, and the etiology and early progression of autism spectrum disorder. It will be argued here that the autism spectrum is an adept model to explore miRNA impact on the cognitive and affective processes that descend from the developing cerebellum.

N-myc is a key switch regulating the proliferation cycle of postnatal cerebellar granule cell progenitors.

N-myc plays an important role in early cerebellar development; however, the role of N-myc in postnatal cerebellar development is still unknown. In this study, inducible and reversible N-myc mouse models (NmycTRE/TRE:tTS and NmycEGFP/TRE:tTS) are used to regulate and track the expression of endogenous N-myc in vivo. Loss of N-myc at the neonatal stage results in reduced proliferation of granule cell precursors (GCPs) and reduced cerebellar volume/mass. Restoration of N-myc expression no later than postnatal day 4 can rescue the cerebellar developmental defect caused by the absence of N-myc after birth. During cerebellar postnatal development, N-myc acts as a key switch, regulating the proliferation cycle of postnatal granule cell progenitors. Loss of N-myc significantly impairs the Sonic hedgehog signalling pathway, and disrupts the expression of cell cycle effectors with a significant reduction of Ccnd2. More importantly, N-myc negatively regulates the expression of microRNA-9 during postnatal cerebellar development. Our findings demonstrate that over-expression of miR-9 can inhibit the proliferation of GCPs. The regulation of these factors by N-myc is at least partly responsible for the switch role of N-myc in the proliferation cycle of GCPs.

Wls provides a new compartmental view of the rhombic lip in mouse cerebellar development.

Math1 is the defining molecule of the cerebellar rhombic lip and Pax6 is downstream in the Math1 pathway. In the present study, we discover that Wntless (Wls) is a novel molecular marker of the cells in the interior face of the rhombic lip throughout normal mouse cerebellar development. Wls expression is found complementary to the expression of Math1 and Pax6, which are localized to the exterior face of the rhombic lip. To determine the interaction between these molecules, we examine the loss-of-Math1 or loss-of-Pax6 in the cerebellum, i.e., the Math1-null and Pax6-null (Sey) mutant cerebella. The presence of Wls-positive cells in the Math1-null rhombic lip indicates that Wls expression is independent of Math1. In the Sey mutant cerebellum, there is an expansion of Wls-expressing cells into regions that are normally colonized by Pax6-expressing cells. The ectopic expression of Wls in the Pax6-null cerebellum suggests a negative interaction between Wls-expressing cells and Pax6-positive cells. These findings suggest that the rhombic lip is dynamically patterned by the expression of Wls, Math1, and Pax6. We also examine five rhombic lip cell markers (Wls, Math1, Pax6, Lmx1a, and Tbr2) to identify four molecularly distinct compartments in the rhombic lip during cerebellar development. The existence of spatial compartmentation in the rhombic lip and the interplay between Wls, Math1, and Pax6 in the rhombic lip provides novel views of early cerebellar development.

Deregulated FGF and homeotic gene expression underlies cerebellar vermis hypoplasia in CHARGE syndrome

Mutations in CHD7 are the major cause of CHARGE syndrome, an autosomal dominant disorder with an estimated prevalence of 1/15,000. We have little understanding of the disruptions in the developmental programme that underpin brain defects associated with this syndrome. Using mouse models, we show that Chd7 haploinsufficiency results in reduced Fgf8 expression in the isthmus organiser (IsO), an embryonic signalling centre that directs early cerebellar development. Consistent with this observation, Chd7 and Fgf8 loss-of-function alleles interact during cerebellar development. CHD7 associates with Otx2 and Gbx2 regulatory elements and altered expression of these homeobox genes implicates CHD7 in the maintenance of cerebellar identity during embryogenesis. Finally, we report cerebellar vermis hypoplasia in 35% of CHARGE syndrome patients with a proven CHD7 mutation. These observations provide key insights into the molecular aetiology of cerebellar defects in CHARGE syndrome and link reduced FGF signalling to cerebellar vermis hypoplasia in a human syndrome. DOI: http://dx.doi.org/10.7554/eLife.01305.001.

Identification of MMP-2 as a novel enhancer of cerebellar granule cell proliferation

During the first postnatal days in the mouse, granule cells (GCs) undergo massive proliferation, which then gradually decreases. Matrix metalloproteinase-2 (MMP-2), a Zn(2+)-dependent proteolytic enzyme, is involved in a wide variety of pathological and physiological pathways. Evidence for a role of this proteinase in cell proliferation is emerging, reporting its involvement in pathological proliferation, as well as during neurogenesis and developmental proliferation of non-CNS tissues. In this study, MMP-2 protein expression was observed within the early postnatal cerebellar cortex, predominantly in Purkinje cells and within the GC proliferative zone, i.e. the superficial external granular layer (EGL). Consistently, the spatiotemporal MMP-2 mRNA and protein profiles highly correlated with the peak of GC precursor (GCP) proliferation and detailed morphometric analyses of MMP-2 deficient cerebella revealed a thinner EGL due to a decreased GCP proliferation. BrdU cumulative experiments, performed to measure the length of different cell cycle phases, further disclosed a transiently prolonged S-phase in MMP-2 deficient GCPs during early cerebellar development. In consequence, MMP-2 deficient animals displayed a transient delay in GC migration towards the IGL. In conclusion, our findings provide important evidence for a role for MMP-2 in neuronal proliferation and cell cycle kinetics in the developing CNS.

Altered gene expression in the emerging cerebellar primordium of Neurog1−/− mice

Expression of the basic helix–loop–helix (bHLH) transcription factor Neurogenin1 (Neurog1) coincides with the emergence of the cerebellum and Neurog1-expressing progenitors are fated to become Purkinje cells and later interneurons. However, the gene regulatory functions of Neurog1 in cerebellar development have not been characterized. We performed a genome-wide analysis of gene expression in the cerebellar primordium of E11.5 Neurog1 null (Neurog1−/−) mice to identify the Neurog1 transcriptome in the emerging cerebellum. This screen identified 117 genes differentially enriched in Neurog1−/− versus control sample sets with a high presence of gene sets enriched for functions in nervous system development. Hierarchical clustering revealed complete stratification of differentially expressed genes based on Neurog1 gene deletion status. In silico analysis of promoter regions identifies high probability Neurog1 regulatory (E-box) binding sites in 94 of the 117 differentially expressed genes and Pax6 binding motifs in 25 of these 94 promoters. Our data provide a framework for investigating Neurog1 transcriptional programs in early cerebellar development and suggest functional Neurog1–Pax6 cross-talk in the activation of downstream targets.