@article{14794, abstract = {Mosaic analysis with double markers (MADM) technology enables the sparse labeling of genetically defined neurons. We present a protocol for time-lapse imaging of cortical projection neuron migration in mice using MADM. We describe steps for the isolation, culturing, and 4D imaging of neuronal dynamics in MADM-labeled brain tissue. While this protocol is compatible with other single-cell labeling methods, the MADM approach provides a genetic platform for the functional assessment of cell-autonomous candidate gene function and the relative contribution of non-cell-autonomous effects. For complete details on the use and execution of this protocol, please refer to Hansen et al. (2022),1 Contreras et al. (2021),2 and Amberg and Hippenmeyer (2021).3}, author = {Hansen, Andi H and Hippenmeyer, Simon}, issn = {2666-1667}, journal = {STAR Protocols}, number = {1}, publisher = {Elsevier}, title = {{Time-lapse imaging of cortical projection neuron migration in mice using mosaic analysis with double markers}}, doi = {10.1016/j.xpro.2023.102795}, volume = {5}, year = {2024}, } @article{10791, abstract = {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.}, author = {Hansen, Andi H and Pauler, Florian and Riedl, Michael and Streicher, Carmen and Heger, Anna-Magdalena and Laukoter, Susanne and Sommer, Christoph M and Nicolas, Armel and Hof, Björn and Tsai, Li Huei and Rülicke, Thomas and Hippenmeyer, Simon}, issn = {2753-149X}, journal = {Oxford Open Neuroscience}, number = {1}, publisher = {Oxford Academic}, title = {{Tissue-wide effects override cell-intrinsic gene function in radial neuron migration}}, doi = {10.1093/oons/kvac009}, volume = {1}, year = {2022}, } @article{8546, abstract = {Brain neurons arise from relatively few progenitors generating an enormous diversity of neuronal types. Nonetheless, a cardinal feature of mammalian brain neurogenesis is thought to be that excitatory and inhibitory neurons derive from separate, spatially segregated progenitors. Whether bi-potential progenitors with an intrinsic capacity to generate both lineages exist and how such a fate decision may be regulated are unknown. Using cerebellar development as a model, we discover that individual progenitors can give rise to both inhibitory and excitatory lineages. Gradations of Notch activity determine the fates of the progenitors and their daughters. Daughters with the highest levels of Notch activity retain the progenitor fate, while intermediate levels of Notch activity generate inhibitory neurons, and daughters with very low levels of Notch signaling adopt the excitatory fate. Therefore, Notch-mediated binary cell fate choice is a mechanism for regulating the ratio of excitatory to inhibitory neurons from common progenitors.}, author = {Zhang, Tingting and Liu, Tengyuan and Mora, Natalia and Guegan, Justine and Bertrand, Mathilde and Contreras, Ximena and Hansen, Andi H and Streicher, Carmen and Anderle, Marica and Danda, Natasha and Tiberi, Luca and Hippenmeyer, Simon and Hassan, Bassem A.}, issn = { 22111247}, journal = {Cell Reports}, number = {10}, publisher = {Elsevier}, title = {{Generation of excitatory and inhibitory neurons from common progenitors via Notch signaling in the cerebellum}}, doi = {10.1016/j.celrep.2021.109208}, volume = {35}, year = {2021}, } @article{9603, abstract = {Mosaic analysis with double markers (MADM) offers one approach to visualize and concomitantly manipulate genetically defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage, single-cell morphology and physiology, genomic imprinting phenotypes, and dissection of cell-autonomous gene functions in vivo in health and disease. Yet, MADM can only be applied to <25% of all mouse genes on select chromosomes to date. To overcome this limitation, we generate transgenic mice with knocked-in MADM cassettes near the centromeres of all 19 autosomes and validate their use across organs. With this resource, >96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond a proof of principle, we apply our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We find striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division.}, author = {Contreras, Ximena and Amberg, Nicole and Davaatseren, Amarbayasgalan and Hansen, Andi H and Sonntag, Johanna and Andersen, Lill and Bernthaler, Tina and Streicher, Carmen and Heger, Anna-Magdalena and Johnson, Randy L. and Schwarz, Lindsay A. and Luo, Liqun and Rülicke, Thomas and Hippenmeyer, Simon}, issn = {22111247}, journal = {Cell Reports}, number = {12}, publisher = {Cell Press}, title = {{A genome-wide library of MADM mice for single-cell genetic mosaic analysis}}, doi = {10.1016/j.celrep.2021.109274}, volume = {35}, year = {2021}, } @phdthesis{9962, abstract = {The brain is one of the largest and most complex organs and it is composed of billions of neurons that communicate together enabling e.g. consciousness. The cerebral cortex is the largest site of neural integration in the central nervous system. Concerted radial migration of newly born cortical projection neurons, from their birthplace to their final position, is a key step in the assembly of the cerebral cortex. The cellular and molecular mechanisms regulating radial neuronal migration in vivo are however still unclear. Recent evidence suggests that distinct signaling cues act cell-autonomously but differentially at certain steps during the overall migration process. Moreover, functional analysis of genetic mosaics (mutant neurons present in wild-type/heterozygote environment) using the MADM (Mosaic Analysis with Double Markers) analyses in comparison to global knockout also indicate a significant degree of non-cell-autonomous and/or community effects in the control of cortical neuron migration. The interactions of cell-intrinsic (cell-autonomous) and cell-extrinsic (non-cell-autonomous) components are largely unknown. In part of this thesis work we established a MADM-based experimental strategy for the quantitative analysis of cell-autonomous gene function versus non-cell-autonomous and/or community effects. The direct comparison of mutant neurons from the genetic mosaic (cell-autonomous) to mutant neurons in the conditional and/or global knockout (cell-autonomous + non-cell-autonomous) allows to quantitatively analyze non-cell-autonomous effects. Such analysis enable the high-resolution analysis of projection neuron migration dynamics in distinct environments with concomitant isolation of genomic and proteomic profiles. Using these experimental paradigms and in combination with computational modeling we show and characterize the nature of non-cell-autonomous effects to coordinate radial neuron migration. Furthermore, this thesis discusses recent developments in neurodevelopment with focus on neuronal polarization and non-cell-autonomous mechanisms in neuronal migration.}, author = {Hansen, Andi H}, issn = {2663-337X}, keywords = {Neuronal migration, Non-cell-autonomous, Cell-autonomous, Neurodevelopmental disease}, pages = {182}, publisher = {Institute of Science and Technology Austria}, title = {{Cell-autonomous gene function and non-cell-autonomous effects in radial projection neuron migration}}, doi = {10.15479/at:ista:9962}, year = {2021}, } @article{7815, abstract = {Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.}, author = {Beattie, Robert J and Streicher, Carmen and Amberg, Nicole and Cheung, Giselle T and Contreras, Ximena and Hansen, Andi H and Hippenmeyer, Simon}, issn = {1940-087X}, journal = {Journal of Visual Experiments}, number = {159}, publisher = {MyJove Corporation}, title = {{Lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (MADM)}}, doi = {10.3791/61147}, year = {2020}, } @article{8162, abstract = {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.}, author = {Laukoter, Susanne and Pauler, Florian and Beattie, Robert J and Amberg, Nicole and Hansen, Andi H and Streicher, Carmen and Penz, Thomas and Bock, Christoph and Hippenmeyer, Simon}, issn = {0896-6273}, journal = {Neuron}, number = {6}, pages = {1160--1179.e9}, publisher = {Elsevier}, title = {{Cell-type specificity of genomic imprinting in cerebral cortex}}, doi = {10.1016/j.neuron.2020.06.031}, volume = {107}, year = {2020}, } @article{8569, abstract = {Concerted radial migration of newly born cortical projection neurons, from their birthplace to their final target lamina, is a key step in the assembly of the cerebral cortex. The cellular and molecular mechanisms regulating the specific sequential steps of radial neuronal migration in vivo are however still unclear, let alone the effects and interactions with the extracellular environment. In any in vivo context, cells will always be exposed to a complex extracellular environment consisting of (1) secreted factors acting as potential signaling cues, (2) the extracellular matrix, and (3) other cells providing cell–cell interaction through receptors and/or direct physical stimuli. Most studies so far have described and focused mainly on intrinsic cell-autonomous gene functions in neuronal migration but there is accumulating evidence that non-cell-autonomous-, local-, systemic-, and/or whole tissue-wide effects substantially contribute to the regulation of radial neuronal migration. These non-cell-autonomous effects may differentially affect cortical neuron migration in distinct cellular environments. However, the cellular and molecular natures of such non-cell-autonomous mechanisms are mostly unknown. Furthermore, physical forces due to collective migration and/or community effects (i.e., interactions with surrounding cells) may play important roles in neocortical projection neuron migration. In this concise review, we first outline distinct models of non-cell-autonomous interactions of cortical projection neurons along their radial migration trajectory during development. We then summarize experimental assays and platforms that can be utilized to visualize and potentially probe non-cell-autonomous mechanisms. Lastly, we define key questions to address in the future.}, author = {Hansen, Andi H and Hippenmeyer, Simon}, issn = {2296-634X}, journal = {Frontiers in Cell and Developmental Biology}, number = {9}, publisher = {Frontiers}, title = {{Non-cell-autonomous mechanisms in radial projection neuron migration in the developing cerebral cortex}}, doi = {10.3389/fcell.2020.574382}, volume = {8}, year = {2020}, }