Lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (MADM)

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

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

A mathematical insight into cell labelling experiments for clonal analysis

Studying the progression of the proliferative and differentiative patterns of neural stem cells at the individual cell-level is crucial to the understanding of cortex development and how the disruption of such patterns can lead to malformations and neurodevelopmental diseases. However, our understanding of the precise lineage progression program at single cell resolution is still incomplete due to the technical variations in lineage tracing approaches. One of the key challenges involves developing a robust theoretical framework in which we can integrate experimental observations and introduce correction factors to obtain a reliable and representative description of the temporal modulation of proliferation and differentiation. In order to obtain more conclusive insights we carry out virtual clonal analysis using mathematical modelling and compare our results against experimental data. Using a dataset obtained with Mosaic Analysis with Double Markers, we illustrate how the theoretical description can be exploited to interpret and reconcile the disparity between virtual and experimental results

A genome-wide library of MADM mice for single-cell genetic mosaic analysis

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 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

Deterministic progenitor behavior and unitary production of neurons in the neocortex.

Radial glial progenitors (RGPs) are responsible for producing nearly all neocortical neurons. To gain insight into the patterns of RGP division and neuron production, we quantitatively analyzed excitatory neuron genesis in the mouse neocortex using Mosaic Analysis with Double Markers, which provides single-cell resolution of progenitor division patterns and potential in vivo. We found that RGPs progress through a coherent program in which their proliferative potential diminishes in a predictable manner. Upon entry into the neurogenic phase, individual RGPs produce ?8-9 neurons distributed in both deep and superficial layers, indicating a unitary output in neuronal production. Removal of OTX1, a transcription factor transiently expressed in RGPs, results in both deep- and superficial-layer neuron loss and a reduction in neuronal unit size. Moreover, ?1/6 of neurogenic RGPs proceed to produce glia. These results suggest that progenitor behavior and histogenesis in the mammalian neocortex conform to a remarkably orderly and deterministic program.This work was supported by the European Union (FP7-CIG618444 to S.H.), the Simons Foundation (to S.-H.S.), NIH grants (R01DA024681 and R01MH101382 to S.-H.S., R01NS050835 to L.L., and T32HD060600 to L.H.), the Wellcome Trust (098357/Z/12/Z to B.D.S.) and the Human Frontier Science Program (RGP0053 to S.-H.S., S.H., B.D.S. and K.H.). L.L. is an investigator of the Howard Hughes Medical Institute.This is the final version. It was first published by Elsevier at http://www.cell.com/cell/abstract/S0092-8674%2814%2901315-

Tissue-wide genetic and cellular landscape shapes the execution of sequential PRC2 functions in neural stem cell lineage progression

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

Clonally related forebrain interneurons disperse broadly across both functional areas and structural boundaries

The medial ganglionic eminence (MGE) gives rise to the majority of mouse forebrain interneurons. Here, we examine the lineage relationship among MGE-derived interneurons using a replication-defective retroviral library containing a highly diverse set of DNA barcodes. Recovering the barcodes from the mature progeny of infected progenitor cells enabled us to unambiguously determine their respective lineal relationship. We found that clonal dispersion occurs across large areas of the brain and is not restricted by anatomical divisions. As such, sibling interneurons can populate the cortex, hippocampus striatum, and globus pallidus. The majority of interneurons appeared to be generated from asymmetric divisions of MGE progenitor cells, followed by symmetric divisions within the subventricular zone. Altogether, our findings uncover that lineage relationships do not appear to determine interneuron allocation to particular regions. As such, it is likely that clonally related interneurons have considerable flexibility as to the particular forebrain circuits to which they can contribute

El (des)gobierno en la pandemia del COVID-19 y las implicaciones psicosociales: disciplinamiento, sujeción y subjetividad

Objetivo: analizar las implicaciones psicosociales derivadas de la pandemia de la COVID-19, relatadas en el servicio online, desde la perspectiva de los conceptos de biopoder, biopolítica y gubernamentalidad de Michel Foucault. Método: investigación documental cualitativa, con análisis de prontuarios de usuarios atendidos en chat de escucha terapéutica, entre abril y octubre de 2020. Resultados: los datos fueron organizados en dos temas: Gubernamentalidad en la pandemia de la COVID-19 y la producción de implicaciones psicosociales de la ansiedad y el miedo y Disciplinas y sujeciones en la pandemia de la COVID-19: subjetividades marcadas por la tristeza y la angustia. El primero demuestra que el “arte de gobernar” en Brasil produjo inestabilidades e incertidumbres que influyeron en la producción de miedo a la contaminación/muerte/y no acceso al tratamiento y ansiedad. En el segundo tema, podemos ver cómo se combinan el control disciplinario y la regulación biopolítica. En Brasil, país sumamente desigual, se han producido subjetividades y subjetividades marcadas por la angustia, sentimientos de desánimo y tristeza. Conclusión: los procesos de exclusión se profundizaron en la pandemia, con el ejercicio de una biopolítica que precariza la vida y produce sufrimiento psíquico.Objetivo: analisar as implicações psicossociais decorrentes da pandemia da COVID-19, relatadas em atendimento online, pela ótica dos conceitos de biopoder, biopolítica e de governamentalidade de Michel Foucault. Método: pesquisa qualitativa do tipo documental, com a análise dos registros de prontuários de usuários atendidos em um chat de escuta terapêutica, entre abril e outubro de 2020. Resultados: os dados foram organizados em duas temáticas: Governamentalidade na pandemia de COVID-19 e a produção de implicações psicossociais de ansiedade e medo e Disciplinarizações e sujeições na pandemia de COVID-19: subjetividades marcadas pela tristeza e angústia. A primeira demonstra que a “arte de governar” no Brasil produziu instabilidades e incertezas que influenciaram na produção do medo da contaminação/morte/e não acesso ao tratamento e ansiedade. Na segunda temática, percebe-se como o controle disciplinar e a regulamentação biopolítica se combinam. No Brasil, um país extremamente desigual, tem-se produzido sujeição e subjetividades marcadas pela angústia, sentimentos de desânimo e tristeza. Conclusão: os processos excludentes foram aprofundados na pandemia, com exercício de uma biopolítica que precariza a vida e produz sofrimento psíquico.Objective: to analyze the psychosocial implications arising from the COVID-19 pandemic, reported in online service, from the perspective of Michel Foucault’s concepts of biopower, biopolitics and governmentality. Method: qualitative documental research, with analysis of medical records of users assisted in a therapeutic listening chat, between April and October 2020. Results: the data were organized into two themes: Governmentality in the COVID-19 pandemic and the production of psychosocial implications of anxiety and fear and Discipline and subjection in the COVID-19 pandemic: subjectivities marked by sadness and anguish. The first demonstrates that the “art of governing” in Brazil produced instabilities and uncertainties that influenced the production of fear of contamination/death/and non-access to treatment and anxiety. In the second theme, we can see how disciplinary control and biopolitical regulation are combined. In Brazil, an extremely unequal country, subjectivity and subjectivities marked by anguish, feelings of discouragement and sadness have been produced. Conclusion: the exclusionary processes were deepened in the pandemic, with the exercise of a biopolitics that makes life precarious and produces psychological distress

Multipotent progenitors instruct ontogeny of the superior colliculus

The superior colliculus (SC) in the mammalian midbrain is essential for multisensory integration, attention, and complex behavior (Basso and May, 2017; Cang et al., 2018). The mature SC cytoarchitecture is organized into distinct laminae and composed of a rich variety of neuronal and glial cell types (Ayupe et al., 2023; Edwards et al., 1986; May, 2006; Xie et al., 2021; Zeisel et al., 2018). Precise execution of the developmental programs regulating the generation of SC cell-type diversity is essential, because deficits due to genetic mutations have been associated with neurodevelopmental diseases and SC dysfunction (Jure, 2018; McFadyen et al., 2020). However, the fundamentals directing the ontogeny of the SC are not well understood. Here we pursued systematic lineage tracing at the single progenitor cell level in order to decipher the principles instructing the generation of cell-type diversity in the SC. We combined in silico lineage reconstruction with a novel genetic MADM (Mosaic Analysis with Double Markers)-CloneSeq approach. MADM-CloneSeq enables the unequivocal delineation of cell lineages in situ, and cell identity based on global transcriptome, of individual clonally-related cells. Our systematic reconstructions of cell lineages revealed that all neuronal cell types in SC emerge from local progenitors without any extrinsic source. Strikingly, individual SC progenitors are exceptionally multipotent with the capacity to produce all known excitatory and inhibitory neuron types of the prospective mature SC, with individual clonal units showing no pre-defined composition. At the molecular level we identified an essential role for PTEN signaling in establishing appropriate proportions of specific inhibitory and excitatory neuron types. Collectively, our findings demonstrate that individual multipotent progenitors generate the full spectrum of excitatory and inhibitory neuron types in the developing SC, providing a novel framework for the emergence of cell-type diversity and thus the ontogeny of the mammalian SC