@article{14683, abstract = {Mosaic analysis with double markers (MADM) technology enables the generation of genetic mosaic tissue in mice and high-resolution phenotyping at the individual cell level. Here, we present a protocol for isolating MADM-labeled cells with high yield for downstream molecular analyses using fluorescence-activated cell sorting (FACS). We describe steps for generating MADM-labeled mice, perfusion, single-cell suspension, and debris removal. We then detail procedures for cell sorting by FACS and downstream analysis. This protocol is suitable for embryonic to adult mice. For complete details on the use and execution of this protocol, please refer to Contreras et al. (2021).1}, author = {Amberg, Nicole and Cheung, Giselle T and Hippenmeyer, Simon}, issn = {2666-1667}, journal = {STAR Protocols}, keywords = {General Immunology and Microbiology, General Biochemistry, Genetics and Molecular Biology, General Neuroscience}, number = {1}, publisher = {Elsevier}, title = {{Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry}}, doi = {10.1016/j.xpro.2023.102771}, volume = {5}, year = {2023}, } @inbook{14757, abstract = {The cerebral cortex is comprised of a vast cell-type diversity sequentially generated by cortical progenitor cells. Faithful progenitor lineage progression requires the tight orchestration of distinct molecular and cellular mechanisms regulating proper progenitor proliferation behavior and differentiation. Correct execution of developmental programs involves a complex interplay of cell intrinsic and tissue-wide mechanisms. Many studies over the past decades have been able to determine a plethora of genes critically involved in cortical development. However, only a few made use of genetic paradigms with sparse and global gene deletion to probe cell-autonomous vs. tissue-wide contribution. In this chapter, we will elaborate on the importance of dissecting the cell-autonomous and tissue-wide mechanisms to gain a precise understanding of gene function during radial glial progenitor lineage progression.}, author = {Villalba Requena, Ana and Amberg, Nicole and Hippenmeyer, Simon}, booktitle = {Neocortical Neurogenesis in Development and Evolution}, editor = {Huttner, Wieland}, pages = {169--191}, publisher = {Wiley}, title = {{Interplay of Cell‐autonomous Gene Function and Tissue‐wide Mechanisms Regulating Radial Glial Progenitor Lineage Progression}}, doi = {10.1002/9781119860914.ch10}, year = {2023}, } @article{12802, abstract = {Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction.}, author = {Knaus, Lisa and Basilico, Bernadette and Malzl, Daniel and Gerykova Bujalkova, Maria and Smogavec, Mateja and Schwarz, Lena A. and Gorkiewicz, Sarah and Amberg, Nicole and Pauler, Florian and Knittl-Frank, Christian and Tassinari, Marianna and Maulide, Nuno and Rülicke, Thomas and Menche, Jörg and Hippenmeyer, Simon and Novarino, Gaia}, issn = {0092-8674}, journal = {Cell}, keywords = {General Biochemistry, Genetics and Molecular Biology}, number = {9}, pages = {1950--1967.e25}, publisher = {Elsevier}, title = {{Large neutral amino acid levels tune perinatal neuronal excitability and survival}}, doi = {10.1016/j.cell.2023.02.037}, volume = {186}, year = {2023}, } @article{14257, abstract = {Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease.}, author = {Michalska, Julia M and Lyudchik, Julia and Velicky, Philipp and Korinkova, Hana and Watson, Jake and Cenameri, Alban and Sommer, Christoph M and Amberg, Nicole and Venturino, Alessandro and Roessler, Karl and Czech, Thomas and Höftberger, Romana and Siegert, Sandra and Novarino, Gaia and Jonas, Peter M and Danzl, Johann G}, issn = {1546-1696}, journal = {Nature Biotechnology}, publisher = {Springer Nature}, title = {{Imaging brain tissue architecture across millimeter to nanometer scales}}, doi = {10.1038/s41587-023-01911-8}, year = {2023}, } @article{11336, abstract = {The generation of a correctly-sized cerebral cortex with all-embracing neuronal and glial cell-type diversity critically depends on faithful radial glial progenitor (RGP) cell proliferation/differentiation programs. Temporal RGP lineage progression is regulated by Polycomb Repressive Complex 2 (PRC2) and loss of PRC2 activity results in severe neurogenesis defects and microcephaly. How PRC2-dependent gene expression instructs RGP lineage progression is unknown. Here we utilize Mosaic Analysis with Double Markers (MADM)-based single cell technology and demonstrate that PRC2 is not cell-autonomously required in neurogenic RGPs but rather acts at the global tissue-wide level. Conversely, cortical astrocyte production and maturation is cell-autonomously controlled by PRC2-dependent transcriptional regulation. We thus reveal highly distinct and sequential PRC2 functions in RGP lineage progression that are dependent on complex interplays between intrinsic and tissue-wide properties. In a broader context our results imply a critical role for the genetic and cellular niche environment in neural stem cell behavior.}, author = {Amberg, Nicole and Pauler, Florian and Streicher, Carmen and Hippenmeyer, Simon}, issn = {2375-2548}, journal = {Science Advances}, number = {44}, publisher = {American Association for the Advancement of Science}, title = {{Tissue-wide genetic and cellular landscape shapes the execution of sequential PRC2 functions in neural stem cell lineage progression}}, doi = {10.1126/sciadv.abq1263}, volume = {8}, year = {2022}, } @article{12282, abstract = {From a simple thought to a multicellular movement}, author = {Amberg, Nicole and Stouffer, Melissa A and Vercellino, Irene}, issn = {1477-9137}, journal = {Journal of Cell Science}, number = {8}, publisher = {The Company of Biologists}, title = {{Operation STEM fatale – how an equity, diversity and inclusion initiative has brought us to reflect on the current challenges in cell biology and science as a whole}}, doi = {10.1242/jcs.260017}, volume = {135}, year = {2022}, } @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}, } @article{10321, abstract = {Mosaic analysis with double markers (MADM) technology enables the generation of genetic mosaic tissue in mice. MADM enables concomitant fluorescent cell labeling and introduction of a mutation of a gene of interest with single-cell resolution. This protocol highlights major steps for the generation of genetic mosaic tissue and the isolation and processing of respective tissues for downstream histological analysis. For complete details on the use and execution of this protocol, please refer to Contreras et al. (2021).}, author = {Amberg, Nicole and Hippenmeyer, Simon}, issn = {2666-1667}, journal = {STAR Protocols}, number = {4}, publisher = {Cell Press}, title = {{Genetic mosaic dissection of candidate genes in mice using mosaic analysis with double markers}}, doi = {10.1016/j.xpro.2021.100939}, volume = {2}, year = {2021}, } @article{8978, abstract = {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).}, author = {Laukoter, Susanne and Amberg, Nicole and Pauler, Florian and Hippenmeyer, Simon}, issn = {2666-1667}, journal = {STAR Protocols}, number = {3}, publisher = {Elsevier}, title = {{Generation and isolation of single cells from mouse brain with mosaic analysis with double markers-induced uniparental chromosome disomy}}, doi = {10.1016/j.xpro.2020.100215}, volume = {1}, year = {2020}, } @article{7253, abstract = {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.}, author = {Laukoter, Susanne and Beattie, Robert J and Pauler, Florian and Amberg, Nicole and Nakayama, Keiichi I. and Hippenmeyer, Simon}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Imprinted Cdkn1c genomic locus cell-autonomously promotes cell survival in cerebral cortex development}}, doi = {10.1038/s41467-019-14077-2}, volume = {11}, year = {2020}, } @article{8093, abstract = {Background: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment. Methods: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo. Results: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system. Conclusion: We propose that the chemokine axis CCL20–CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.}, author = {Hippe, Andreas and Braun, Stephan Alexander and Oláh, Péter and Gerber, Peter Arne and Schorr, Anne and Seeliger, Stephan and Holtz, Stephanie and Jannasch, Katharina and Pivarcsi, Andor and Buhren, Bettina and Schrumpf, Holger and Kislat, Andreas and Bünemann, Erich and Steinhoff, Martin and Fischer, Jens and Lira, Sérgio A. and Boukamp, Petra and Hevezi, Peter and Stoecklein, Nikolas Hendrik and Hoffmann, Thomas and Alves, Frauke and Sleeman, Jonathan and Bauer, Thomas and Klufa, Jörg and Amberg, Nicole and Sibilia, Maria and Zlotnik, Albert and Müller-Homey, Anja and Homey, Bernhard}, issn = {1532-1827}, journal = {British Journal of Cancer}, pages = {942--954}, publisher = {Springer Nature}, title = {{EGFR/Ras-induced CCL20 production modulates the tumour microenvironment}}, doi = {10.1038/s41416-020-0943-2}, volume = {123}, 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{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{6455, abstract = {During corticogenesis, distinct subtypes of neurons are sequentially born from ventricular zone progenitors. How these cells are molecularly temporally patterned is poorly understood. We used single-cell RNA sequencing at high temporal resolution to trace the lineage of the molecular identities of successive generations of apical progenitors (APs) and their daughter neurons in mouse embryos. We identified a core set of evolutionarily conserved, temporally patterned genes that drive APs from internally driven to more exteroceptive states. We found that the Polycomb repressor complex 2 (PRC2) epigenetically regulates AP temporal progression. Embryonic age–dependent AP molecular states are transmitted to their progeny as successive ground states, onto which essentially conserved early postmitotic differentiation programs are applied, and are complemented by later-occurring environment-dependent signals. Thus, epigenetically regulated temporal molecular birthmarks present in progenitors act in their postmitotic progeny to seed adult neuronal diversity.}, author = {Telley, L and Agirman, G and Prados, J and Amberg, Nicole and Fièvre, S and Oberst, P and Bartolini, G and Vitali, I and Cadilhac, C and Hippenmeyer, Simon and Nguyen, L and Dayer, A and Jabaudon, D}, issn = {1095-9203}, journal = {Science}, number = {6440}, publisher = {AAAS}, title = {{Temporal patterning of apical progenitors and their daughter neurons in the developing neocortex}}, doi = {10.1126/science.aav2522}, volume = {364}, year = {2019}, } @article{6451, abstract = {Epidermal growth factor receptor (EGFR) signaling controls skin development and homeostasis inmice and humans, and its deficiency causes severe skin inflammation, which might affect epidermalstem cell behavior. Here, we describe the inflammation-independent effects of EGFR deficiency dur-ing skin morphogenesis and in adult hair follicle stem cells. Expression and alternative splicing analysisof RNA sequencing data from interfollicular epidermis and outer root sheath indicate that EGFR con-trols genes involved in epidermal differentiation and also in centrosome function, DNA damage, cellcycle, and apoptosis. Genetic experiments employingp53deletion in EGFR-deficient epidermis revealthat EGFR signaling exhibitsp53-dependent functions in proliferative epidermal compartments, aswell asp53-independent functions in differentiated hair shaft keratinocytes. Loss of EGFR leads toabsence of LEF1 protein specifically in the innermost epithelial hair layers, resulting in disorganizationof medulla cells. Thus, our results uncover important spatial and temporal features of cell-autonomousEGFR functions in the epidermis.}, author = {Amberg, Nicole and Sotiropoulou, Panagiota A. and Heller, Gerwin and Lichtenberger, Beate M. and Holcmann, Martin and Camurdanoglu, Bahar and Baykuscheva-Gentscheva, Temenuschka and Blanpain, Cedric and Sibilia, Maria}, issn = {2589-0042}, journal = {iScience}, pages = {243--256}, publisher = {Elsevier}, title = {{EGFR controls hair shaft differentiation in a p53-independent manner}}, doi = {10.1016/j.isci.2019.04.018}, volume = {15}, year = {2019}, } @article{27, abstract = {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 (NSC) lineages. The vast majority of cortical projection neurons and certain classes of glial cells are generated by radial glial progenitor cells (RGPs) in a highly orchestrated manner. Recent studies employing single cell analysis and clonal lineage tracing suggest that NSC and RGP 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. This article is protected by copyright. All rights reserved.}, author = {Amberg, Nicole and Laukoter, Susanne and Hippenmeyer, Simon}, journal = {Journal of Neurochemistry}, number = {1}, pages = {12--26}, publisher = {Wiley}, title = {{Epigenetic cues modulating the generation of cell type diversity in the cerebral cortex}}, doi = {10.1111/jnc.14601}, volume = {149}, year = {2019}, }