Tissue-Wide Effects Override Cell-Intrinsic Gene Function in Radial Neuron Migration.

The mammalian neocortex is composed of diverse neuronal and glial cell classes that broadly arrange in six distinct laminae. Cortical layers emerge during development and defects in the developmental programs that orchestrate cortical lamination are associated with neurodevelopmental diseases. The developmental principle of cortical layer formation depends on concerted radial projection neuron migration, from their birthplace to their final target position. Radial migration occurs in defined sequential steps, regulated by a large array of signaling pathways. However, based on genetic loss-of-function experiments, most studies have thus far focused on the role of cell-autonomous gene function. Yet, cortical neuron migration in situ is a complex process and migrating neurons traverse along diverse cellular compartments and environments. The role of tissue-wide properties and genetic state in radial neuron migration is however not clear. Here we utilized mosaic analysis with double markers (MADM) technology to either sparsely or globally delete gene function, followed by quantitative single-cell phenotyping. The MADM-based gene ablation paradigms in combination with computational modeling demonstrated that global tissue-wide effects predominate cell-autonomous gene function albeit in a gene-specific manner. Our results thus suggest that the genetic landscape in a tissue critically affects the overall migration phenotype of individual cortical projection neurons. In a broader context, our findings imply that global tissue-wide effects represent an essential component of the underlying etiology associated with focal malformations of cortical development in particular, and neurological diseases in general.

Inducible uniparental chromosome disomy to probe genomic imprinting at single-cell level in brain and beyond.

Genomic imprinting is an epigenetic mechanism that results in parental allele-specific expression of ~1% of all genes in mouse and human. Imprinted genes are key developmental regulators and play pivotal roles in many biological processes such as nutrient transfer from the mother to offspring and neuronal development. Imprinted genes are also involved in human disease, including neurodevelopmental disorders, and often occur in clusters that are regulated by a common imprint control region (ICR). In extra-embryonic tissues ICRs can act over large distances, with the largest surrounding Igf2r spanning over 10 million base-pairs. Besides classical imprinted expression that shows near exclusive maternal or paternal expression, widespread biased imprinted expression has been identified mainly in brain. In this review we discuss recent developments mapping cell type specific imprinted expression in extra-embryonic tissues and neocortex in the mouse. We highlight the advantages of using an inducible uniparental chromosome disomy (UPD) system to generate cells carrying either two maternal or two paternal copies of a specific chromosome to analyze the functional consequences of genomic imprinting. Mosaic Analysis with Double Markers (MADM) allows fluorescent labeling and concomitant induction of UPD sparsely in specific cell types, and thus to over-express or suppress all imprinted genes on that chromosome. To illustrate the utility of this technique, we explain how MADM-induced UPD revealed new insights about the function of the well-studied Cdkn1c imprinted gene, and how MADM-induced UPDs led to identification of highly cell type specific phenotypes related to perturbed imprinted expression in the mouse neocortex. Finally, we give an outlook on how MADM could be used to probe cell type specific imprinted expression in other tissues in mouse, particularly in extra-embryonic tissues.

Generation and isolation of single cells from mouse brain with mosaic analysis with double markers-induced uniparental chromosome disomy.

Mosaic analysis with double markers (MADM) technology enables concomitant fluorescent cell labeling and induction of uniparental chromosome disomy (UPD) with single-cell resolution. In UPD, imprinted genes are either overexpressed 2-fold or are not expressed. Here, the MADM platform is utilized to probe imprinting phenotypes at the transcriptional level. This protocol highlights major steps for the generation and isolation of projection neurons and astrocytes with MADM-induced UPD from mouse cerebral cortex for downstream single-cell and low-input sample RNA-sequencing experiments. For complete details on the use and execution of this protocol, please refer to Laukoter et al. (2020b).

Cell-Type Specificity of Genomic Imprinting in Cerebral Cortex.

In mammalian genomes, a subset of genes is regulated by genomic imprinting, resulting in silencing of one parental allele. Imprinting is essential for cerebral cortex development, but prevalence and functional impact in individual cells is unclear. Here, we determined allelic expression in cortical cell types and established a quantitative platform to interrogate imprinting in single cells. We created cells with uniparental chromosome disomy (UPD) containing two copies of either the maternal or the paternal chromosome; hence, imprinted genes will be 2-fold overexpressed or not expressed. By genetic labeling of UPD, we determined cellular phenotypes and transcriptional responses to deregulated imprinted gene expression at unprecedented single-cell resolution. We discovered an unexpected degree of cell-type specificity and a novel function of imprinting in the regulation of cortical astrocyte survival. More generally, our results suggest functional relevance of imprinted gene expression in glial astrocyte lineage and thus for generating cortical cell-type diversity.

Imprinted Cdkn1c genomic locus cell-autonomously promotes cell survival in cerebral cortex development.

The cyclin-dependent kinase inhibitor p57KIP2 is encoded by the imprinted Cdkn1c locus, exhibits maternal expression, and is essential for cerebral cortex development. How Cdkn1c regulates corticogenesis is however not clear. To this end we employ Mosaic Analysis with Double Markers (MADM) technology to genetically dissect Cdkn1c gene function in corticogenesis at single cell resolution. We find that the previously described growth-inhibitory Cdkn1c function is a non-cell-autonomous one, acting on the whole organism. In contrast we reveal a growth-promoting cell-autonomous Cdkn1c function which at the mechanistic level mediates radial glial progenitor cell and nascent projection neuron survival. Strikingly, the growth-promoting function of Cdkn1c is highly dosage sensitive but not subject to genomic imprinting. Collectively, our results suggest that the Cdkn1c locus regulates cortical development through distinct cell-autonomous and non-cell-autonomous mechanisms. More generally, our study highlights the importance to probe the relative contributions of cell intrinsic gene function and tissue-wide mechanisms to the overall phenotype.

Epigenetic cues modulating the generation of cell-type diversity in the cerebral cortex.

The cerebral cortex is composed of a large variety of distinct cell-types including projection neurons, interneurons, and glial cells which emerge from distinct neural stem cell lineages. The vast majority of cortical projection neurons and certain classes of glial cells are generated by radial glial progenitor cells in a highly orchestrated manner. Recent studies employing single cell analysis and clonal lineage tracing suggest that neural stem cell and radial glial progenitor lineage progression are regulated in a profound deterministic manner. In this review we focus on recent advances based mainly on correlative phenotypic data emerging from functional genetic studies in mice. We establish hypotheses to test in future research and outline a conceptual framework how epigenetic cues modulate the generation of cell-type diversity during cortical development.

Mosaic Analysis with Double Markers Reveals Distinct Sequential Functions of Lgl1 in Neural Stem Cells.

The concerted production of neurons and glia by neural stem cells (NSCs) is essential for neural circuit assembly. In the developing cerebral cortex, radial glia progenitors (RGPs) generate nearly all neocortical neurons and certain glia lineages. RGP proliferation behavior shows a high degree of non-stochasticity, thus a deterministic characteristic of neuron and glia production. However, the cellular and molecular mechanisms controlling RGP behavior and proliferation dynamics in neurogenesis and glia generation remain unknown. By using mosaic analysis with double markers (MADM)-based genetic paradigms enabling the sparse and global knockout with unprecedented single-cell resolution, we identified Lgl1 as a critical regulatory component. We uncover Lgl1-dependent tissue-wide community effects required for embryonic cortical neurogenesis and novel cell-autonomous Lgl1 functions controlling RGP-mediated glia genesis and postnatal NSC behavior. These results suggest that NSC-mediated neuron and glia production is tightly regulated through the concerted interplay of sequential Lgl1-dependent global and cell intrinsic mechanisms.

Differences in T cell cytotoxicity and cell death mechanisms between progressive multifocal leukoencephalopathy, herpes simplex virus encephalitis and cytomegalovirus encephalitis.

During the appearance of human immunodeficiency virus infection in the 1980 and the 1990s, progressive multifocal leukoencephalopathy (PML), a viral encephalitis induced by the JC virus, was the leading opportunistic brain infection. As a result of the use of modern immunomodulatory compounds such as Natalizumab and Rituximab, the number of patients with PML is once again increasing. Despite the presence of PML over decades, little is known regarding the mechanisms leading to death of infected cells and the role the immune system plays in this process. Here we compared the presence of inflammatory T cells and the targeting of infected cells by cytotoxic T cells in PML, herpes simplex virus encephalitis (HSVE) and cytomegalovirus encephalitis (CMVE). In addition, we analyzed cell death mechanisms in infected cells in these encephalitides. Our results show that large numbers of inflammatory cytotoxic T cells are present in PML lesions. Whereas in HSVE and CMVE, single or multiple appositions of CD8+ or granzyme-B+ T cells to infected cells are found, in PML such appositions are significantly less apparent. Analysis of apoptotic pathways by markers such as activated caspase-3, caspase-6, poly(ADP-ribose) polymerase-1 (PARP-1) and apoptosis-inducing factor (AIF) showed upregulation of caspase-3 and loss of caspase-6 from mitochondria in CMVE and HSVE infected cells. Infected oligodendrocytes in PML did not upregulate activated caspase-3 but instead showed translocation of PARP-1 from nucleus to cytoplasm and AIF from mitochondria to nucleus. These findings suggest that in HSVE and CMVE, cells die by caspase-mediated apoptosis induced by cytotoxic T cells. In PML, on the other hand, infected cells are not eliminated by the immune system but seem to die by virus-induced PARP and AIF translocation in a type of cell death defined as parthanatos.