Human Fetal Cerebellum Research Articles

A human fetal cerebellar map of the late second trimester reveals developmental molecular characteristics and abnormality in trisomy 21

Our understanding of human fetal cerebellum development during the late second trimester, a critical period for the generation of astrocytes, oligodendrocytes, and unipolar brush cells (UBCs), remains limited. Here, we performed single-cell RNA sequencing (scRNA-seq) in human fetal cerebellum samples from gestational weeks (GWs) 18-25. We find that proliferating UBC progenitors distribute in the subventricular zone of the rhombic lip (RLSVZ) near white matter (WM), forming a layer structure. We also delineate two trajectories from astrogenic radial glia (ARGs) to Bergmann glial progenitors (BGPs) and recognize oligodendrogenic radial glia (ORGs) as one source of primitive oligodendrocyte progenitor cells (PriOPCs). Additionally, our scRNA-seq analysis of the trisomy 21 fetal cerebellum at this stage reveals abnormal upregulated genes in pathways such as the cell adhesion pathway and focal adhesion pathway, which potentially promote neuronal differentiation. Overall, our research provides valuable insights into normal and abnormal development of the human fetal cerebellum.

Laminar Architecture and Morphometry of Developing Human Fetal Cerebellum

Abstract Background: One of the organs in the human fetus that starts to differentiate extremely early and keeps doing so throughout the postnatal period is the cerebellum. The goal of this study was to create a nomogram using the chronological events that were occurring and the relationship with progressing gestational age. Granule cells and Purkinje cells play a crucial role in the normal development of the cerebellum. Any disruption at the cellular level can lead to abnormal migration of these cells, potentially resulting in hypoplasia of the cerebellar vermis. Methodology: Tissue specimens from 60 human fetuses were studied for histological changes after being grouped into four from 13th week to 32nd week. Fetuses with any neurological deficit were excluded from the study. Different layers were identified, and the thickness of each layer was noted. Results: The first two groups had three layers, but the composition was different in both. The marginal and mantle layers in the second group disappeared completely. The third group showed presence of five layers, with addition of lamina dissecans, appearing for a transient period. The fourth group had four layers. The thickness of external granular layer and internal granular layer (IGL) increased throughout, except that the IGL made an appearance from the second group only. Lamina dissecans appeared, causing a significant shift between 23 and 27 weeks. The fetus has a four-layered structure near term, with an external granular layer persisting throughout the early postnatal stage of life. Progression of pregnancy was positively connected with the molecular and Purkinje cell layer (PCL) thickness. Conclusion: Progression of pregnancy was positively connected with the molecular and PCL thickness. Age determination and neuropsychiatric disorders of the developing fetus may benefit from such a link.

EPEN-08. MULTI-OMICS PROFILES REVEAL UNIQUE DEFINING MOLECULAR FEATURES OF POSTERIOR FOSSA EPENDYMOMA SUBTYPE A FROM INFANTS

Abstract BACKGROUND Ependymoma is a heterogeneous brain cancer with multiple subgroups based on locations and molecular profiles. Tumours of the methylation class, posterior fossa group A (PF-EPN-A) represent the most common and aggressive neoplasms and have a poor outcome. PF-EPN-A tumours in infants (under three years of age) are understudied; this project aimed to characterise these infant tumours. METHODS We collected DNA methylation (n=570) and gene expression (n=56) profiles of PF-EPN-A tumours, and single-cell RNA sequencing data of human fetal cerebellum from published studies. RESULTS Analysis of copy number profiles derived from DNA methylation arrays showed that infants with PF-EPN-A have relatively quiescent genomes when compared to children aged four to eighteen years (mean fraction of changes in infants (n=300) = 0.018 and children (n=270) = 0.055; p-value < 2.2 × 10-16). No recurrent focal amplifications or losses were detected, however, copy number loss involving chromosome arms, 12q (12%), 7q (9.7%), 5p (8.3%), and 16p (6%) were relatively common in infants, while 1q gain, 6q loss, and 22q loss were more frequent in children. When we stratified PF-EPN-A based on copy number alterations, infants with altered genomes were associated with poor outcomes compared to infants with stable genomes (fraction of changes = 0), which in turn showed poor overall survival compared to children with stable genomes. Gene set enrichment analysis using data from microarrays showed that infant tumours were specifically enriched with genes involved in the extracellular matrix organization, interferon gamma signaling, neuronal system, autophagy, and signaling by Interleukins, VEGF and PDGF. Integration with single-cell data from the human fetal cerebellum revealed that infants in PF-EPN-A had significantly higher proportions of glial progenitor cells and decreased granule cell progenitor subpopulations. CONCLUSIONS Together, our study identifies distinct genetic and transcriptional programs among infants in PF-EPN-A tumours, representing opportunities to refine future treatment.

MiR-124-3p and miR-194-5p regulation of the PI3K/AKT pathway via ROR2 in medulloblastoma progression

Medulloblastoma (MB), a prevalent pediatric central nervous system tumor, is influenced by microRNAs (miRNAs) that impact tumor initiation and progression. However, the specific involvement of miRNAs in MB tumorigenesis remains unclear. Using single-cell RNA sequencing, we identified ROR2 expression in normal human fetal cerebellum. Subsequent analyses, including immunofluorescence, quantitative real-time PCR (qRT-PCR), and Western blot, assessed ROR2 expression in MB tissues and cell lines. We investigated miR-124-3p and miR-194-5p and their regulatory role in ROR2 expression through the dual-luciferase reporter, qRT-PCR, and western blot assays. Mechanistic insights were gained through functional assays exploring the impact of miR-124-3p, miR-194-5p, and ROR2 on MB growth in vitro and in vivo. We observed significantly reduced miR-124-3p and miR-194-5p expression and elevated ROR2 expression in MB tissues and cell lines. High ROR2 expression inversely correlated with overall survival in WNT and SHH subgroups of MB patients. Functionally, overexpressing miR-124-3p and miR-194-5p and inhibiting ROR2 suppressed in vitro malignant transformation and in vivo tumorigenicity. Mechanistically, miR-124-3p and miR-194-5p synergistically regulated the ROR2/PI3K/Akt pathway, influencing MB progression. Our findings indicate that miR-124-3p and miR-194-5p function as tumor suppressors, inhibiting MB progression via the ROR2/PI3K/Akt axis, suggesting a key mechanism and therapeutic targets for MB patients.

Single-cell multi-omics analysis of lineage development and spatial organization in the human fetal cerebellum

Human cerebellum encompasses numerous neurons, exhibiting a distinct developmental paradigm from cerebrum. Here we conducted scRNA-seq, scATAC-seq and spatial transcriptomic analyses of fetal samples from gestational week (GW) 13 to 18 to explore the emergence of cellular diversity and developmental programs in the developing human cerebellum. We identified transitory granule cell progenitors that are conserved across species. Special patterns in both granule cells and Purkinje cells were dissected multidimensionally. Species-specific gene expression patterns of cerebellar lobes were characterized and we found that PARM1 exhibited inconsistent distribution in human and mouse granule cells. A novel cluster of potential neuroepithelium at the rhombic lip was identified. We also resolved various subtypes of Purkinje cells and unipolar brush cells and revealed gene regulatory networks controlling their diversification. Therefore, our study offers a valuable multi-omics landscape of human fetal cerebellum and advances our understanding of development and spatial organization of human cerebellum.

A Transitional Cell Population Is Enriched in Aggressive Human Medulloblastoma.

A transitional progenitor population, enhanced in aggressive medulloblastoma, was identified in human fetal cerebellum.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Histomorphometry of the cortical layers and the dentate nucleus of the human fetal cerebellum

ObjectivesThis study was aimed at determining the histomorphometry of the cerebellar cortical laminae and the dentate nucleus of the human fetal cerebellum; the number and shape of the neurons; and the gestational age of appearance of the cerebellar folia, white matter and arbor vitae cerebelli. MethodsMicroscopic sections of the human fetal cerebellum stained with hematoxylin and eosin and Bielschowsky silver stain were studied. ResultsThe thickness of the cortical laminae of the human fetal cerebellum varied among gestational weeks as follows: external granular layer: 36.06 ± 9.36–50.05 ± 34.06 μm, molecular layer: 32.76 ± 17.16–52 ± 28.6 μm, Purkinje cell layer: 9.36 ± 6.8–15.6 ± 4.68 μm and internal granular layer: 66.65 ± 24.42–146.63 ± 47.79 μm. Similarly, the number of neurons per field of view at 1000X under a compound microscope varied among gestational weeks as follows: external granular layer: 89.92 ± 42–142.84 ± 50, molecular layer: 15 ± 12.5–25 ± 8.25, Purkinje cell layer: 3.5 ± 1–5 ± 2.5 and internal granular layer: 98.5 ± 69.75–224 ± 47.White matter in the fetal cerebellum was already present at the age of 12th gestational week, whereas cerebellar folia appeared at 16–20 gestational weeks. Arbor vitae cerebelli and the dentate nucleus became conspicuous after the 20th gestational week. Fetal neurons were round except for Purkinje cells. ConclusionsThe thickness and neuronal counts of the human fetal cerebellar cortical layers and the measurements of the dentate nucleus along with other histomorphological features varied with gestational age from the 12th week of gestation until birth.

MEDB-78. Unified rhombic lip origins of Group 3 and Group 4 medulloblastoma

Identification and characterization of lineage-specific beginnings of distinct medulloblastoma (MB) subgroups is a fundamental challenge in the field. Genetically engineered mouse models and cross-species transcriptomics have provided mounting evidence of discrete, subgroup-specific developmental origins. Likewise, murine single-cell transcriptional atlases of cerebellar development have recently provided further clues into MB subgroup origins, particularly for poorly defined Group 3 and Group 4-MB. However, initial studies were underpowered to characterize rare populations and lacked robust validation, resulting in incomplete findings. Herein, we leveraged a large harmonized murine cerebellar atlas, targeted lineage enrichment, and integrative multi-omic strategies to deeply dissect MB origins. Isolation of spatially and temporally discrete developmental trajectories of key glutamatergic lineages born out of the murine upper rhombic lip provided an enhanced reference for mapping MB subgroup origins, especially for Group 3 and Group 4-MB. However, human-specific anatomic and cellular complexity, particularly within the rhombic lip germinal zone complicated murine-derived inferences. Further tumor-normal integrations using a novel single-cell atlas of the human fetal cerebellum, companioned with laser-capture micro-dissected transcriptional and epigenetic datasets, reinforced developmental insights extracted from candidate murine cerebellar lineages. Characterization of compartment-specific transcriptional signatures identified in the human upper rhombic lip implicated convergent cellular correlates of Group 3 and Group 4-MB, suggestive of a common developmental trajectory underlying their ancestry. Systematic imaging review and 3D summarization of a large clinical trial series of patient tumors, coupled with our advanced insights into developmental signatures, substantiated subgroup-specific tumor location patterns observed at diagnosis. Together, our results strongly implicate a common lineage trajectory of the upper rhombic lip as the probable origin of Group 3 and Group 4-MB. These important findings provide unprecedented opportunities to explore context-dependent mechanisms of MB pathogenesis and will foster generation of improved preclinical models that more faithfully recapitulate tumor biology.

EPCO-26. INTEGRATIVE MULTI-OMICS IDENTIFIES CONVERGING DEVELOPMENTAL ORIGINS OF DISTINCT MEDULLOBLASTOMA SUBGROUPS

Abstract Understanding the interplay between normal development and tumorigenesis, including the identification and characterization of lineage-specific origins of MB, is a fundamental challenge in the field. Recent studies have highlighted novel associations between biologically distinct MB subgroups and diverse murine cerebellar lineages via cross-species single-cell transcriptomics. Specifically, Group 4-MB correlated with the unipolar brush cell lineage and Group 3-MB resembled Nestin+ stem cells of the early cerebellum. However, these analyses were hampered by low resolution due to the sparsity of pertinent cerebellar cell types and the cross-species nature of the approach. Herein, we profoundly expand the depth of these rare developmental populations in the murine cerebellum using a combination of lineage tracing and integrative multi-omics. Isolation and enrichment of spatially and temporally unique developmental trajectories of key rhombic lip-derived glutamatergic lineages provided an enhanced reference for mapping MB subgroups based on molecular overlap, especially for poorly defined Group 3- and Group 4-MB. Further comparisons to a novel single-cell atlas of the human fetal cerebellum, companioned with laser-capture microdissected transcriptional and epigenetic datasets, reinforced developmental insights extracted from the mouse. Characterization of compartment-specific transcriptional programs and co-expression networks identified in the human upper rhombic lip implicated convergent cellular correlates of Group 3- and Group 4-MB, suggestive of a common developmental link. Together, our results strongly implicate developmental lineages of the upper rhombic lip as the probable origins of poorly defined Group 3- and Group 4-MB. These important findings will shape future efforts to accurately model the biological heterogeneity underlying these subgroups and provide unprecedented opportunities to explore their cellular and mechanistic basis.

Spatial and cell type transcriptional landscape of human cerebellar development.

The human neonatal cerebellum is a fourth of its adult size, yet contains the blueprint required to integrate environmental cues with developing motor, cognitive, and emotional skills into adulthood. Although mature cerebellar neuroanatomy is well studied, understanding its developmental origins is limited. Here, we systematically mapped the molecular, cellular, and spatial composition of human fetal cerebellum by combining laser capture microscopy and SPLiT-seq single-nucleus transcriptomics. We profiled functionally distinct regions and gene expression dynamics within cell types and across development. The resulting cell atlas demonstrates that the molecular organization of the cerebellar anlage recapitulates cytoarchitecturally distinct regions and developmentally transient cell types that are distinct from the mouse cerebellum. By mapping genes dominant for pediatric and adult neurological disorders onto our dataset, we identify relevant cell types underlying disease mechanisms. These data provide a resource for probing the cellular basis of human cerebellar development and disease.

Morphometric development of the human fetal cerebellum during the early second trimester

The protracted nature of development makes the cerebellum vulnerable to a broad spectrum of pathologic conditions, especially during the early fetal period. This study aims to characterize normal cerebellar growth in human fetuses during the early second trimester. We manually segmented the fetal cerebellum using 7.0-T high-resolution MR images obtained in 35 specimens with gestational ages ranging from 15 to 22 weeks. Volume measurements and shape analysis were performed to quantitatively evaluate global and regional cerebellar growth. The absolute volume of the fetal cerebellum showed a quadratic growth with increasing gestational age, while the pattern of relative volume changes revealed that the cerebellum grew at a greater pace than the cerebrum after 17 gestational weeks. Shape analysis was used to examine the distinctive development of subregions of the cerebellum. The extreme lateral portions of both cerebellar hemispheres showed the lowest rate of growth. The anterior lobe grew faster than most of the posterior lobe. These findings expand our understanding of the early growth pattern of the human cerebellum and could be further used to assess the developmental conditions of the fetal brain.

Characterization of cancer stem cells and primary cilia in medulloblastoma.

Medulloblastoma, a tumor of the cerebellum, is the most common pediatric central nervous system malignancy. These tumors are etiologically linked to mutations in the Sonic hedgehog (Shh) pathway, which signals through the primary, non-motile cilium. The growth of these aggressive tumors relies on self-renewal of tumor-propagating cells known as cancer stem cells (CSCs). Previous reports have implicated CD133-expressing cells as CSCs in brain tumors, while those expressing CD15 have been shown to propagate medulloblastoma. Here, we demonstrate that CD133+ and CD15+ cells are distinct medulloblastoma populations. CD15+ cells comprise approximately 0.5-1% of total human medulloblastoma cells, display CSC properties in culture and are detected in the Smoothened A1 transgenic mouse model of medulloblastoma. Additionally, we report on a medulloblastoma patient with enriched CD15+ cells in recurrent vs primary medulloblastoma. We also demonstrate that human medulloblastoma cells critically rely on establishment of primary cilia to drive Shh-mediated cell division. Primary cilia are found in external granule cells of human fetal cerebellum and in 12/14 medulloblastoma samples. Yet, CD15+ medulloblastoma cells lack primary cilia, suggesting that this CSC population signals independently of Shh. These results are important when considering the effects of current and prospective treatment modalities on medulloblastoma CSC populations.

Multiple microRNAs regulate human FOXP2 gene expression by targeting sequences in its 3' untranslated region.

BackgroundMutations in the human FOXP2 gene cause speech and language impairments. The FOXP2 protein is a transcription factor that regulates the expression of many downstream genes, which may have important roles in nervous system development and function. An adequate amount of functional FOXP2 protein is thought to be critical for the proper development of the neural circuitry underlying speech and language. However, how FOXP2 gene expression is regulated is not clearly understood. The FOXP2 mRNA has an approximately 4-kb-long 3′ untranslated region (3′ UTR), twice as long as its protein coding region, indicating that FOXP2 can be regulated by microRNAs (miRNAs).FindingsWe identified multiple miRNAs that regulate the expression of the human FOXP2 gene using sequence analysis and in vitro cell systems. Focusing on let-7a, miR-9, and miR-129-5p, three brain-enriched miRNAs, we show that these miRNAs regulate human FOXP2 expression in a dosage-dependent manner and target specific sequences in the FOXP2 3′ UTR. We further show that these three miRNAs are expressed in the cerebellum of the human fetal brain, where FOXP2 is known to be expressed.ConclusionsOur results reveal novel regulatory functions of the human FOXP2 3′ UTR sequence and regulatory interactions between multiple miRNAs and the human FOXP2 gene. The expression of let-7a, miR-9, and miR-129-5p in the human fetal cerebellum is consistent with their roles in regulating FOXP2 expression during early cerebellum development. These results suggest that various genetic and environmental factors may contribute to speech and language development and related neural developmental disorders via the miRNA-FOXP2 regulatory network.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-014-0071-0) contains supplementary material, which is available to authorized users.

Deconstruction of Medulloblastoma Cellular Heterogeneity Reveals Differences between the Most Highly Invasive and Self-Renewing Phenotypes

Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor. Major research efforts have focused on characterizing and targeting putative brain tumor stem or propagating cell populations from the tumor mass. However, less is known about the relationship between these cells and highly invasive MB cells that evade current therapies. Here, we dissected MB cellular heterogeneity and directly compared invasion and self-renewal. Analysis of higher versus lower self-renewing tumor spheres and stationary versus migrating adherent MB cells revealed differential expression of the cell surface markers CD271 [p75 neurotrophin receptor (p75NTR)] and CD133. Cell sorting demonstrated that CD271 selects for subpopulations with a higher capacity for self-renewal, whereas CD133 selects for cells exhibiting increased invasion in vitro. CD271 expression is higher in human fetal cerebellum and primary samples of the Shh MB molecular variant and lower in the more aggressive, invasive group 3 and 4 subgroups. Global gene expression analysis of higher versus lower self-renewing MB tumor spheres revealed down-regulation of a cell movement transcription program in the higher self-renewing state and a novel potential role for axon guidance signaling in MB-propagating cells. We have identified a cell surface signature based on CD133/CD271 expression that selects for MB cells with a higher self-renewal potential or invasive capacity in vitro. Our study underscores a previously unappreciated role for CD271 in selecting for MB cell phenotypes and suggests that successful treatment of pediatric brain tumors requires concomitant targeting of a spectrum of transitioning self-renewing and highly infiltrative cell subpopulations.

3D Morphometric Analysis of Human Fetal Cerebellar Development

To date, growth of the human fetal cerebellum has been estimated primarily from linear measurements from ultrasound and 2D magnetic resonance imaging (MRI). In this study, we use 3D analytical methods to develop normative growth trajectories for the cerebellum in utero. We measured cerebellar volume, linear dimensions, and local surface curvature from 3D reconstructed MRI of the human fetal brain (N = 46). We found that cerebellar volume increased approximately 7-fold from 20 to 31 gestational weeks. The better fit of the exponential curve (R (2) = 0.96) compared to the linear curve (R (2) = 0.92) indicated acceleration in growth. Within-subject cerebellar and cerebral volumes were highly correlated (R (2) = 0.94), though the cerebellar percentage of total brain volume increased from approximately 2.4% to 3.7% (R (2) = 0.63). Right and left hemispheric volumes did not significantly differ. Transcerebellar diameter, vermal height, and vermal anterior to posterior diameter increased significantly at constant rates. From the local curvature analysis, we found that expansion along the inferior and superior aspects of the hemispheres resulted in decreased convexity, which is likely due to the physical constraints of the dura surrounding the cerebellum and the adjacent brainstem. The paired decrease in convexity along the inferior vermis and increased convexity of the medial hemisphere represents development of the paravermian fissure, which becomes more visible during this period. In this 3D morphometric analysis of the human fetal cerebellum, we have shown that cerebellar growth is accelerating at a greater pace than the cerebrum and described how cerebellar growth impacts the shape of the structure.

Development of the human fetal cerebellum in the second trimester: a post mortem magnetic resonance imaging evaluation

The cerebellum is one of the most important structures in the posterior cranial fossa, but the characterization of its development by magnetic resonance imaging (MRI) is incomplete. We scanned 40 fetuses that had no morphological brain disorder at 14-22 weeks of gestation using 7.0 T MRI. Amira 4.1 software was used to determine morphological parameters of the fetal cerebellum, which included the cerebellar volume (CV), transverse cerebellar diameter (TCD), and the length and width of the vermis. The relationship between these measurements and gestational age (GA) was analysed. We found that the primary fissure was visible at week 14 of gestation. From week 16, the prepyramidal fissure, the secondary fissure and the dentate nucleus could be identified. The posterolateral fissure and the fourth ventricle were recognized at week 17, whereas the tentorium of the cerebellum was visible at week 20. The relationships between GA and CV, TCD, and the width and length of the vermis were described adequately by second-order polynomial regression curves. The ratios between TCD and vermis length and between TCD and vermis width decreased with GA. These results show that 7.0 T MRI can show the trajectory of cerebellar development clearly. They increase our understanding of normal cerebellar development in the fetus, and will facilitate the diagnosis of pathological intrauterine changes in the cerebellum.

Application of Chip-Based Nanoelectrospray Ion Trap Mass Spectrometry to Compositional and Structural Analysis of Gangliosides in Human Fetal Cerebellum

Two native ganglioside mixtures from normal human fetal cerebellum in the 15th (Cc15) and 40th (Cc40) gestational week were subjected to NanoMate high capacity ion trap (HCT) mass spectrometric (MS) and collision induced dissociation (CID) tandem MS (MS2) analysis under thoroughly optimized experimental conditionns. An total of 56 different species were identified in Cc15 and 54 in Cc40. By employing CID MS2 molecular ions, related GD1 (d18:1/20:0) and GM2 (d18:1/19:0) species were structurally characterized in a high throughput mode. The method provided elevated ionization efficiency, high speed of analysis, almost 100% reproducibility at sample consumption per experiment situated in the femtomole range.

Diffusion tensor imaging in the developing human cerebellum with histologic correlation

Diffusion tensor imaging was performed on 24 freshly aborted human fetuses with gestational age ranging from 20 to 37 weeks to observe age-related fractional anisotropy changes in cerebellar cortex and cerebellar white matter. Quantitative immunohistochemical analysis was performed for glial fibrillary acidic protein in each fetus molecular layer of cerebellar cortex and myelin basic protein expression was quantified in myelinated areas of the middle cerebellar peduncles. The cerebellar cortical fractional anisotropy reached its peak value at 28 weeks, and then decreased gradually until 37 weeks. The time course of glial fibrillary acidic protein expression paralleled that of fractional anisotropy in the cerebellar cortex from 20 weeks of gestation upto the gestational age at which the fractional anisotropy reached its peak value (28 weeks). In the middle cerebellar peduncles, the fractional anisotropy increased continuously upto 37 weeks of gestational age and showed a significant positive correlation with myelin basic protein immunostained fibers. The fractional anisotropy quantification can be used to assess the migrational and maturation changes during the development of the human fetal cerebellum supported by the immunohistochemical analysis.

Primary and secondary transcriptional effects in the developing human Down syndrome brain and heart

Microarray analysis of transcript levels in fetal cerebellum and heart tissues of Down syndrome patients showed a disruption only of chromosome 21 gene expression.