@unpublished{21291, abstract = {The complexity and specificity of movement in vertebrates is driven by a rich diversity of spinal motor and interneuron cell types. During development, eleven spinal cord progenitor domains generate an equivalent number of cardinal neuron types. How progenitor domains, individual progenitors, and post-mitotic diversity relate is still unknown. We performed high-resolution, single-progenitor cell lineage tracing in the embryonic mouse spinal cord using mosaic analysis with double markers (MADM). Our quantitative study of lineage progression revealed that spinal cord progenitors undergo highly variable numbers of proliferative, neurogenic, and gliogenic cell divisions. The nascent clonally-related neurons migrate radially over large distances, span the dorsoventral axis, and even cross the midline, demonstrating striking bilaterality. Molecular and morphometric analysis indicate high levels of progenitor multipotency, with an individual progenitor capable of producing several molecularly and morphologically distinct neuron types, as well as astrocytes. These findings redefine spinal cord development as a process in which lineage variability — rather than rigid progenitor identity — drives the generation of cellular diversity.}, author = {Gobeil, Sophie A and Da Silveira Neto, Francisco and Silvestrelli, Giulia and Smits, Matthijs Geert and Streicher, Carmen and Cheung, Giselle T and Hippenmeyer, Simon and Sweeney, Lora Beatrice Jaeger}, booktitle = {bioRxiv}, title = {{Lineage origin of spinal cord cell type diversity}}, doi = {10.64898/2026.02.12.705305}, year = {2026}, } @article{21948, abstract = {The cerebral cortex comprises diverse neuron and glial cell types generated by radial glial progenitors (RGPs) during development. Although RGPs broadly differentiate according to temporally and spatially regulated molecular logics, the lineage hierarchies linking individual progenitors to defined cell (sub)types are not well understood. Clone-resolved transcriptomics, combining molecular barcoding and single-cell RNA sequencing, allow high-resolution lineage tracing at the single-clone/cell level across different species and models. In this mini-review, we synthesize recent advances in this field, uncovering unexpected lineage relationships in the developing brain, with a particular focus on the cerebral cortex. We further highlight new insights into species-specific differences in the developmental programs generating cell-type diversity, linking changes in clonal architecture to lineage diversification during cortical evolution.}, author = {Varela Martínez, Irene and Pipicelli, Fabrizia and Hippenmeyer, Simon}, issn = {1879-0380}, journal = {Current Opinion in Genetics and Development}, publisher = {Elsevier}, title = {{Tracing cell lineages in the developing brain: Insights from mosaic analysis and clone-resolved transcriptomics}}, doi = {10.1016/j.gde.2026.102487}, volume = {99}, year = {2026}, } @unpublished{21963, abstract = {The cerebral cortex consists of immense numbers of neuronal and glial cell-types derived from radial glial progenitor (RGP) cells. How RGPs generate appropriate quantities of distinct cortical cell-types to safeguard a brain of correct size, is not well understood. However, genetic aberration in human, including mutations in PTEN, lead to cortical malformation such as macrocephaly, albeit with unknown etiology. Here we utilized Mosaic Analysis with Double Markers (MADM)-based clonal analysis and single cell phenotyping to decipher the role of Pten in neurogenic and gliogenic RGP lineage progression during cortical ontogeny. While neurogenic RGP lineage progression and projection neuron production was moderately altered in the absence of Pten, cortical astrocyte production was drastically increased. Through genetic epistasis experiments we show that the loss of Pten uncouples astrocyte generation from essential growth factor signaling hubs, funneling into MAPK. Collectively, our results suggest that Pten regulates RGP lineage progression with distinct sequential functions in cortical projection neurogenesis and astrocyte production to ensure the emergence of a correctly-sized cerebral cortex.}, author = {Miranda, Osvaldo and Contreras, Ximena and Pauler, Florian and Davaatseren, Amarbayasgalan and Amberg, Nicole and Streicher, Carmen and Villalba Requena, Ana and Heger, Anna-Magdalena and Marie, Corentine and Hassan, Bassem A. and Rülicke, Thomas and Hippenmeyer, Simon}, booktitle = {bioRxiv}, title = {{Pten orchestrates neurogenic radial glia lineage progression and tunes neocortical astrocyte production}}, doi = {10.64898/2026.05.01.722191}, year = {2026}, } @article{19718, abstract = {The cerebral cortex is arguably the most complex organ in humans. The cortical architecture is characterized by a remarkable diversity of neuronal and glial cell types that make up its neuronal circuits. Following a precise temporally ordered program, radial glia progenitor (RGP) cells generate all cortical excitatory projection neurons and glial cell-types. Cortical excitatory projection neurons are produced either directly or via intermediate progenitors, through indirect neurogenesis. How the extensive cortical cell-type diversity is generated during cortex development remains, however, a fundamental open question. How do RGPs quantitatively and qualitatively generate all the neocortical neurons? How does direct and indirect neurogenesis contribute to the establishment of neuronal and lineage heterogeneity? Whether RGPs represent a homogeneous and/or multipotent progenitor population, or if RGPs consist of heterogeneous groups is currently also not known. In this review, we will summarize the latest findings that contributed to a deeper insight into the above key questions.}, author = {Pipicelli, Fabrizia and Villalba Requena, Ana and Hippenmeyer, Simon}, issn = {0959-4388}, journal = {Current Opinion in Neurobiology}, publisher = {Elsevier}, title = {{How radial glia progenitor lineages generate cell-type diversity in the developing cerebral cortex}}, doi = {10.1016/j.conb.2025.103046}, volume = {93}, year = {2025}, } @unpublished{19762, abstract = {The cerebral cortex must contain the appropriate numbers of neurons in each layer to acquire its proper functional organization. Accordingly, neurogenesis requires precise regulation along development. Cortical neurons are made either directly by Radial Glia Cells (RGCs) that self- consume, or indirectly from RGCs via Intermediate Progenitor Cells (IPCs) and largely preserving the RGC pool. According to the standing model of cortical development, Direct Neurogenesis predominates at early stages of development, and progressively shifts to Indirect Neurogenesis, which predominates at late stages. However, neurogenesis at early stages should be compatible with RGC amplification, and neurogenesis at late stages needs to involve RGC consumption, which seems in conflict with the standing model. Here we studied the modes of neurogenesis along cortical development using multiple approaches, including birthdating, live imaging and MADM clone labeling. Contrary to the established dogma, our data show that Indirect Neurogenesis clearly predominates at early developmental stages, gradually shifting to Direct Neurogenesis at late stages. These findings challenge the current model of cortical neurogenesis, and prompt a re-evaluation of previous and ongoing work about the genetic and molecular mechanisms regulating this process.}, author = {Cárdenas, Adrián and Çelik, Irem and Espinós, Alexandre and Streicher, Carmen and López-González, Lara and del-Valle-Anton, Lucia and Fernández, Virginia and Amin, Salma and Negri, Enrico and Ortuño, Eduardo Fernández and Hippenmeyer, Simon and Borrell, Víctor}, booktitle = {bioRxiv}, title = {{Early indirect neurogenesis transitions to late direct neurogenesis in mouse cerebral cortex development}}, doi = {10.1101/2025.05.22.655488}, year = {2025}, } @phdthesis{20737, author = {Casado Polanco, Raquel}, isbn = {978-3-99078-072-5}, issn = {2663-337X}, keywords = {NOTCH, radial glial progenitor, lineage progression, cortical development}, pages = {133}, publisher = {Institute of Science and Technology Austria}, title = {{Role of NOTCH signaling in radial glial progenitor lineage progression}}, doi = {10.15479/AT-ISTA-20737}, year = {2025}, } @article{12875, abstract = {The superior colliculus (SC) in the mammalian midbrain is essential for multisensory integration and is composed of a rich diversity of excitatory and inhibitory neurons and glia. However, the developmental principles directing the generation of SC cell-type diversity are not understood. Here, we pursued systematic cell lineage tracing in silico and in vivo, preserving full spatial information, using genetic mosaic analysis with double markers (MADM)-based clonal analysis with single-cell sequencing (MADM-CloneSeq). The analysis of clonally related cell lineages revealed that radial glial progenitors (RGPs) in SC are exceptionally multipotent. Individual resident RGPs have the capacity to produce all excitatory and inhibitory SC neuron types, even at the stage of terminal division. While individual clonal units show no pre-defined cellular composition, the establishment of appropriate relative proportions of distinct neuronal types occurs in a PTEN-dependent manner. Collectively, our findings provide an inaugural framework at the single-RGP/-cell level of the mammalian SC ontogeny.}, author = {Cheung, Giselle T and Pauler, Florian and Koppensteiner, Peter and Krausgruber, Thomas and Streicher, Carmen and Schrammel, Martin and Özgen, Natalie Y and Ivec, Alexis and Bock, Christoph and Shigemoto, Ryuichi and Hippenmeyer, Simon}, issn = {0896-6273}, journal = {Neuron}, number = {2}, pages = {230--246.e11}, publisher = {Elsevier}, title = {{Multipotent progenitors instruct ontogeny of the superior colliculus}}, doi = {10.1016/j.neuron.2023.11.009}, volume = {112}, year = {2024}, } @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 = {2024}, } @article{17187, abstract = {The generation of diverse cell types during development is fundamental to brain functions. We outline a protocol to quantitatively assess the clonal output of individual neural progenitors using mosaic analysis with double markers (MADM) in mice. We first describe steps to acquire and reconstruct adult MADM clones in the superior colliculus. Then we detail analysis pipelines to determine clonal composition and architecture. This protocol enables the buildup of quantitative frameworks of lineage progression with precise spatial resolution in the brain. For complete details on the use and execution of this protocol, please refer to Cheung et al.1}, author = {Cheung, Giselle T and Streicher, Carmen and Hippenmeyer, Simon}, issn = {2666-1667}, journal = {STAR Protocols}, number = {3}, publisher = {Elsevier}, title = {{Protocol for quantitative reconstruction of cell lineage using mosaic analysis with double markers in mice}}, doi = {10.1016/j.xpro.2024.103157}, volume = {5}, year = {2024}, } @article{17232, abstract = {The lineage relationship of clonally-related cells offers important insights into the ontogeny and cytoarchitecture of the brain in health and disease. Here, we provide a protocol to concurrently assess cell lineage relationship and cell-type identity among clonally-related cells in situ. We first describe the preparation and screening of acute brain slices containing clonally-related cells labeled using mosaic analysis with double markers (MADM). We then outline steps to collect RNA from individual cells for downstream applications and cell-type identification using RNA sequencing. For complete details on the use and execution of this protocol, please refer to Cheung et al. 1}, author = {Cheung, Giselle T and Pauler, Florian and Koppensteiner, Peter and Hippenmeyer, Simon}, issn = {2666-1667}, journal = {STAR Protocols}, number = {3}, publisher = {Elsevier}, title = {{Protocol for mapping cell lineage and cell-type identity of clonally-related cells in situ using MADM-CloneSeq}}, doi = {10.1016/j.xpro.2024.103168}, volume = {5}, year = {2024}, } @inbook{17425, abstract = {Mosaic Analysis with Double Markers (MADM) is a powerful genetic method typically used for lineage tracing and to disentangle cell autonomous and tissue-wide roles of candidate genes with single cell resolution. Given the relatively sparse labeling, depending on which of the 19 MADM chromosomes one chooses, the MADM approach represents the perfect opportunity for cell morphology analysis. Various MADM studies include reports of morphological anomalies and phenotypes in the central nervous system (CNS). MADM for any candidate gene can easily incorporate morphological analysis within the experimental workflow. Here, we describe the methods of morphological cell analysis which we developed in the course of diverse recent MADM studies. This chapter will specifically focus on methods to quantify aspects of the morphology of neurons and astrocytes within the CNS, but these methods can broadly be applied to any MADM-labeled cells throughout the entire organism. We will cover two analyses—soma volume and dendrite characterization—of physical characteristics of pyramidal neurons in the somatosensory cortex, and two analyses—volume and Sholl analysis—of astrocyte morphology.}, author = {Miranda, Osvaldo and Cheung, Giselle T and Hippenmeyer, Simon}, booktitle = {Neuronal Morphogenesis}, editor = {Toyooka, Kazuhito}, isbn = {9781071639689}, issn = {1940-6029}, pages = {283--299}, publisher = {Springer Nature}, title = {{Morphological Analysis of Neurons and Glia Using Mosaic Analysis with Double Markers}}, doi = {10.1007/978-1-0716-3969-6_19}, volume = {2831}, year = {2024}, } @article{12679, abstract = {How to generate a brain of correct size and with appropriate cell-type diversity during development is a major question in Neuroscience. In the developing neocortex, radial glial progenitor (RGP) cells are the main neural stem cells that produce cortical excitatory projection neurons, glial cells, and establish the prospective postnatal stem cell niche in the lateral ventricles. RGPs follow a tightly orchestrated developmental program that when disrupted can result in severe cortical malformations such as microcephaly and megalencephaly. The precise cellular and molecular mechanisms instructing faithful RGP lineage progression are however not well understood. This review will summarize recent conceptual advances that contribute to our understanding of the general principles of RGP lineage progression.}, author = {Hippenmeyer, Simon}, issn = {0959-4388}, journal = {Current Opinion in Neurobiology}, keywords = {General Neuroscience}, number = {4}, publisher = {Elsevier}, title = {{Principles of neural stem cell lineage progression: Insights from developing cerebral cortex}}, doi = {10.1016/j.conb.2023.102695}, volume = {79}, year = {2023}, } @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{9073, abstract = {The sensory and cognitive abilities of the mammalian neocortex are underpinned by intricate columnar and laminar circuits formed from an array of diverse neuronal populations. One approach to determining how interactions between these circuit components give rise to complex behavior is to investigate the rules by which cortical circuits are formed and acquire functionality during development. This review summarizes recent research on the development of the neocortex, from genetic determination in neural stem cells through to the dynamic role that specific neuronal populations play in the earliest circuits of neocortex, and how they contribute to emergent function and cognition. While many of these endeavors take advantage of model systems, consideration will also be given to advances in our understanding of activity in nascent human circuits. Such cross-species perspective is imperative when investigating the mechanisms underlying the dysfunction of early neocortical circuits in neurodevelopmental disorders, so that one can identify targets amenable to therapeutic intervention.}, author = {Hanganu-Opatz, Ileana L. and Butt, Simon J. B. and Hippenmeyer, Simon and De Marco García, Natalia V. and Cardin, Jessica A. and Voytek, Bradley and Muotri, Alysson R.}, issn = {1529-2401}, journal = {The Journal of Neuroscience}, keywords = {General Neuroscience}, number = {5}, pages = {813--822}, publisher = {Society for Neuroscience}, title = {{The logic of developing neocortical circuits in health and disease}}, doi = {10.1523/jneurosci.1655-20.2020}, volume = {41}, 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}, }