Developmental and evolutionary dynamics of cis-regulatory elements in mouse cerebellar cells

Organ development is orchestrated by cell- and time-specific gene regulatory networks. In this study, we investigated the regulatory basis of mouse cerebellum development from early neurogenesis to adulthood. By acquiring snATAC-seq profiles for ~90,000 cells spanning eleven stages, we mapped cerebellar cell types and identified candidate cis-regulatory elements (CREs). We detected extensive spatiotemporal heterogeneity among progenitor cells and a gradual divergence in the regulatory programs of cerebellar neurons during differentiation. Comparisons to vertebrate genomes and snATAC-seq profiles for ∼20,000 cerebellar cells from the marsupial opossum revealed a shared decrease in CRE conservation during development and differentiation, but also differences in constraint between cell types. Our work delineates the developmental and evolutionary dynamics of gene regulation in cerebellar cells and provides insights into mammalian organ development.

Single-cell spatial transcriptomic analysis reveals common and divergent features of developing postnatal granule cerebellar cells and medulloblastoma

Background Cerebellar neurogenesis involves the generation of large numbers of cerebellar granule neurons (GNs) throughout development of the cerebellum, a process that involves tight regulation of proliferation and differentiation of granule neuron progenitors (GNPs). A number of transcriptional regulators, including Math1, and the signaling molecules Wnt and Shh have been shown to have important roles in GNP proliferation and differentiation, and deregulation of granule cell development has been reported to be associated with the pathogenesis of medulloblastoma. While the progenitor/differentiation states of cerebellar granule cells have been broadly investigated, a more detailed association between developmental differentiation programs and spatial gene expression patterns, and how these lead to differential generation of distinct types of medulloblastoma remains poorly understood. Here, we provide a comparative single-cell spatial transcriptomics analysis to better understand the similarities and differences between developing granule and medulloblastoma cells. Results To acquire an enhanced understanding of the precise cellular states of developing cerebellar granule cells, we performed single-cell RNA sequencing of 24,919 murine cerebellar cells from granule neuron-specific reporter mice (Math1-GFP; Dcx-DsRed mice). Our single-cell analysis revealed that there are four major states of developing cerebellar granule cells, including two subsets of granule progenitors and two subsets of differentiating/differentiated granule neurons. Further spatial transcriptomics technology enabled visualization of their spatial locations in cerebellum. In addition, we performed single-cell RNA sequencing of 18,372 cells from Patched+/− mutant mice and found that the transformed granule cells in medulloblastoma closely resembled developing granule neurons of varying differentiation states. However, transformed granule neuron progenitors in medulloblastoma exhibit noticeably less tendency to differentiate compared with cells in normal development. Conclusion In sum, our study revealed the cellular and spatial organization of the detailed states of cerebellar granule cells and provided direct evidence for the similarities and discrepancies between normal cerebellar development and tumorigenesis.

Ankfy1 Is Involved in the Maintenance of Cerebellar Purkinje Cells

Purkinje cells are critical for the function of cerebellum. The degeneration of Purkinje cells leads to defects in motion control. We have found that Purkinje cells specifically express Ankfy1 protein during development and in adult. This protein seems to play minor functions during development as Ankfy1 knockout mice appear normal till adult. However, at 9-month-old, knockout mice showed abnormal cerebellum with reduced vermis size and developed defective motor function. Further investigation demonstrated that the cerebellum of the mutant mouse has lost most of its Purkinje cells, while other cerebellar cells remained largely normal. Our data suggested that the Ankfy1 might be important for the maintenance of cerebellar Purkinje cells.

Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels

Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell–cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca2+ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies.

Tissue and cell-type specific expression of a splice variant in the II-III cytoplasmic loop ofCacna1b

ABSTRACTPresynaptic CaV2.2 (N-type) channels are fundamental for transmitter release across the nervous system. The gene encoding CaV2.2 channels,Cacna1b, contains alternatively spliced exons that originate functionally distinct splice variants (e18a, e24a, e31a and 37a/37b). Alternative splicing of the cassette exon 18a generates two mRNA transcripts (+e18a-Cacna1band Δe18a-Cacna1b). In this study, using novel mouse genetic models and in situ hybridization (BaseScope™), we confirmed that +e18a-Cacna1bsplice variants are expressed in monoaminergic regions of midbrain. We expanded these studies and identified +e18a-Cacna1bmRNA in deep cerebellar cells and spinal cord motor neurons. Furthermore, we determined that +e18a-Cacna1bis enriched in cholecystokinin expressing interneurons. Our results provide key information to understand cell-specific functions of CaV2.2 channels.

The study of the remyelinating effect of leukemia-inhibitor factor and melatonin on the toxic cuprizone model of demyelination of murine cerebellar cells culture in vitro

The cuprizone model of toxic demyelination in vitro is widely used to study the of de- and remyelination in the CNS, as well as to address the issues of finding potential compounds that affect myelination of neuron axons.The aim of the study was to investigate the role of recombinant human leukemia inhibitory factor (rhLIF) and melatonin in remyelination, using the cuprizone demyelination model in vitro.Methods. To study the features of the demyelination and remyelination processes of neuronal axons, the culture of dissociated cerebellar cell culture of the 7-day-old FVB/N lineage mice was used. To detect the myelin sheaths, a histochemical staining with a Sudan Black B was used. To identify oligodendrocytes, immunocytochemical staining of 28-30-old-day cerebellar cells cultures for oligodendrocytes marker Olig2 was performed.Results. The direct effect of the demyelinating factor of cuprizone and remyelination agents (rhLIF and melatonin) on oligodendrocytes in vitro was confirmed. The remyelinating effect of LIF and melatonin on the restoration of myelination processes in dissociated cerebellar cell culture using histochemical and immunocytochemical staining has been revealed.Conclusions. Cuprizone-induced demyelination in vitro is associated with the death of Olig2+ oligodendrocytes and loss of myelin formation. rhLIF and melatonin are prevented the loss of oligodendrocytes and, consequently, reduced the destruction of myelin membranes.

Childhood Cerebellar Tumors Mirror Conserved Fetal Transcriptional Programs

SummaryThe study of the origin and development of cerebellar tumours has been hampered by the complexity and heterogeneity of cerebellar cells that change over the course of development. We used single-cell transcriptomics to study >60,000 cells from the developing murine cerebellum, and show that different molecular subgroups of childhood cerebellar tumors mirror the transcription of cells from distinct, temporally restricted cerebellar lineages. Sonic Hedgehog medulloblastoma transcriptionally mirrors the granule cell hierarchy as expected, whereas Group 3 medulloblastoma resemble Nestin+ve stem cells, Group 4 medulloblastomas resemble unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the prenatal gliogenic progenitor cells. Furthermore, single-cell transcriptomics of human childhood cerebellar tumors demonstrates that many bulk tumors contain a mixed population of cells with divergent differentiation. Our data highlight cerebellar tumors as a disorder of early brain development, and provide a proximate explanation for the peak incidence of cerebellar tumors in early childhood.