Brain development and subsequent brain function are highly sensitive to genetic mutations, which can result in severe neurodevelopmental malformations. Alterations in PTEN signaling cause a spectrum of developmental malformations and neurological diseases including epilepsy. To date, a detailed understanding of the neuropathological underpinnings of PTEN -associated brain malformations, particularly in fetuses, is missing. We have thus investigated a fetal case of hemimegalencephaly (HME), which is a rare disorder characterized by hemispheric overgrowth, developmental delay, and epileptic seizures. Our assessment of the male fetus includes genetic, radiologic, and histologic features and provides a comprehensive characterization of the cellular alterations in HME together with a genotypic correlation. Genetic analyses uncovered that hemispheric overgrowth was caused by a somatic second hit resulting in biallelic PTEN alteration in the affected brain tissue, although the unaffected hemisphere carried the same PTEN variant as the heterozygous germline variant. Based on the latter, we interpret that the PTEN mutation is not a dominant-negative variant. Within the outer subventricular zone of the enlarged cortex, we found small nodular heterotopias, which can be origins of focal epileptic seizures. Cell type-specific marker stainings revealed that the heterotopias consisted exclusively of SATB2 + glutamatergic projection neurons. Altogether, our analyses and findings contribute to a deeper understanding of the pathomechanisms of a novel PTEN variant driving a severe brain malformation.

Bergmann glia (BG) are a specialized glial population essential for cerebellar development, yet their developmental timeline and molecular identity in the human cerebellum remain poorly understood. Here, we combined detailed histopathological analysis with spatial transcriptomics and single-nucleus RNA sequencing to generate a developmental atlas of human cerebellar BG. Histology revealed that BG emerge around 11 postconception weeks (PCW), initially serving as a scaffold for Purkinje cells (PCs) migrating into the PC layer of the cerebellar cortex. Following the establishment of a multilayered PC arrangement, BG form a distinct parallel layer separated from the PCs by the lamina dissecans (LD), with both layers merging in the third trimester. This developmental sequence challenges earlier studies that suggested BG appear late in the third trimester. Comparative histology in mice, ferrets, and marmosets indicates that this trilaminar organization, including the LD, is likely unique to humans. Integration of spatial and single-nucleus transcriptomic datasets identified an ASCL1+ PTF1A+ ventricular zone progenitor cluster giving rise to BG, astrocytes, and oligodendrocytes. Pseudotime analyses delineated three gliogenic lineages and revealed two temporally and transcriptionally distinct BG populations, emerging at 11-12PCW and 17PCW, suggesting multiphasic BG ontogeny. Together, these multimodal data link cellular lineage, spatial organization, and molecular identity of human cerebellar glia, providing a framework for future studies on the role of BG in cerebellar function and their potential contributions to vulnerability in neurodevelopmental disorders.

BACKGROUND:Artificial Intelligence (AI) is rapidly emerging as a transformative tool in medical research and practice. In neuro-oncology, AI may help to enhance diagnostic accuracy and reproducibility, manage complex multi-modal data, and facilitate personalized treatment. METHODS:This review aims to provide an overview of AI applications in the analysis of histopathological and molecular data of brain tumors. RESULTS:Key applications in histopathology include molecular biomarker prediction from H&E stained slides, tumor classification, grading, and prognostication. In molecular pathology, the machine learning-driven DNA methylation-based classification of CNS tumors has already become an integral part of the most recent WHO classification. This framework is continuously refined by ongoing research identifying novel tumor types. Two examples of emerging applications are Stimulated Raman Histology (SRH) and nanopore sequencing. SRH enables an intraoperative AI-powered assessment of the histopathological phenotype. Nanopore sequencing can be used for fast molecular profiling of CNS tumors, including intraoperative methylation-based subtyping. Despite these significant advances, the clinical translation of AI tools faces some challenges, including the limited dataset availability, standardization and representativeness; the lack of robust external validation in many published studies; and the limited model interpretability. These challenges are currently being tackled by efforts to compile multi-institutional pathological datasets and by advances in explainable AI. CONCLUSIONS:AI holds promise for advancing personalized neuro-oncology by improving diagnostic accuracy and accelerating existing workflows. Its potential to democratize access to precision diagnostics hinges on efforts to reduce the costs of digital infrastructure and facilitate specialized training.

Abstract Tumor predisposition syndromes (TPS) contribute to more than 10% of pediatric brain tumors. TPS-associated brain tumors occur in multiple brain regions, with disease severity often linked to tumor location. Thus, one must consider the importance of the microenvironment in cancer initiation. We hypothesize, that distinct microenvironments harbor different vulnerable time points/stem cell identities that undergo oncogenic transformation in a brain region-specific manner. Our experimental model is based on TPS patient-derived iPSCs and brain region-specific organoid cultures to model tumor development in vitro. Our highly reproducible hindbrain and forebrain organoid protocols efficiently induce regional patterning. We focus on the two ultra-rare and highly aggressive TPS termed Constitutional Mismatch Repair Deficiency (CMMRD) and Rhabdoid Tumor Predisposition Syndrome (RTPS) to not only understand critical time windows of oncogenic transformation and the region-derived cues contributing to tumor initiation, but also to dissect TPS-specific mechanisms affecting neural stem cell behavior during development. Within 2 months of culture, we have identified tumor-like lesions in both, forebrain and hindbrain CMMRD-derived organoids. These organoids show a disruption of germinal zone morphology and development of tumor-like lesions, characterized by loss of the apical adherence junction belt, a differentiation block and the formation of hyperproliferative zones. In order to link region-specific physiological outcomes to the (epi)genetic and transcriptomic landscapes, we have performed TSO500 panel sequencing, methylation profiling with nanopore sequencing and snRNAseq. We have uncovered distinct signatures between control and TPS-derived organoids, additional pathological mutations and particular gene expression patterns correlating with oncogenic programs. Our platform offers an unparalleled opportunity to establish a highly relevant translational model for affected patients. Overall, our research seeks to improve the diagnostic and therapeutic options for brain tumor patients by extracting novel biomarkers with predictive value for the clinics.

SUMMARY 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.

Embryonal tumor with multilayered rosettes (ETMR) is a pediatric brain tumor with dismal prognosis. Characteristic alterations of the chromosome 19 microRNA cluster (C19MC) are observed in most ETMR; however, the ramifications of C19MC activation and the complex cellular architecture of ETMR remain understudied. Here we analyze 11 ETMR samples from patients using single-cell transcriptomics and multiplexed spatial imaging. We reveal a spatially distinct cellular hierarchy that spans highly proliferative neural stem-like cells and more differentiated neuron-like cells. C19MC is predominantly expressed in stem-like cells and controls a transcriptional network governing stemness and lineage commitment, as resolved by genome-wide analysis of microRNA-mRNA binding. Systematic analysis of receptor-ligand interactions between malignant cell types reveals fibroblast growth factor receptor and Notch signaling as oncogenic pathways that can be successfully targeted in preclinical models and in one patient with ETMR. Our study provides fundamental insights into ETMR pathobiology and a powerful rationale for more effective targeted therapies.

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

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.

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.

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.

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 use 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.

As a cell biologist, I, Nicole Amberg, have always been fascinated by cell type diversity in nature and how ‘form meets function’ for distinct cell populations. However, when looking at academia, I was discouraged to see that the system does not reflect nature's diverse and variable shapes. Instead, academic institutions (and society as a whole) still lack homogenous access to education, opportunities and chances, resulting in strong imbalances in representation of gender, ethnicity and ultimately and most importantly, mindsets. With a keen sense for equality, I thus set out to become an advocate for progressive change, believing that change can only be implemented by action. I teamed up with several colleagues and founded ‘The STEM fatale Initiative’ (http://stem-fatale.com; @STEM_fatale_ on Twitter; Fig. 1) in order to determine the professional, societal, structural and personal factors impacting women's careers in STEM, and to then propose strategies to improve the current situation. Among the ‘STEM fatale’ participants, I recruited fellow cell biologist Melissa Stouffer and structural biologist Irene Vercellino, partners in marathon training and long conversations on the current state of equity, diversity and inclusion (EDI) in life science, STEM and society at large.Since ‘STEM fatale’ was created and has operated, as a result of the current imbalance in EDI in cell biology, as well as in other scientific disciplines, the three of us (Nicole, Melissa and Irene) decided to take the opportunity in this Essay to report on what we have learned about EDI through our own experiences, mainly focusing on the role of women.As a whole, we have been part of 11 labs in six different countries spread across two continents, forming a personal idea of the status of women in STEM. Additionally, thanks to the ‘STEM fatale’ and via direct interviews with various faculty members at the Institute of Science and Technology Austria (ISTA) – Professors Carrie Bernecky, Tom Henzinger, Martin Loose and Gaia Novarino – we've obtained the perspectives of successful women, but also men, on the topic. Therefore, just like a cell biologist investigates biological phenomena by a plethora of techniques, we have employed interviews, literature and personal experience to address EDI.Taken together, our methods allowed us to identify relevant patterns in the issues faced by women in STEM and present possible solutions to leave a message of hope for a brighter, more equal future. One of the most important contributors to (the lack of) EDI, recurrently encountered in our experiences and reports from others, is unconscious bias. As defined by the neurobiologist Prof. Jennifer Raymond, “Unconscious biases are mental habits that tend to dominate our gut reactions” (e.g. young women are not worth hiring and promoting because they will end up quitting their job once they get pregnant), but importantly “we also have more rational decision processes, which compete with our biases for control of behaviour. Just as one can overcome physical habits […], one can suppress undesirable mental habits such as gender bias through deliberate, conscious strategies” (Raymond, 2013).Since unconscious biases are instinctively present in humans, there is no way to eliminate them, but we can and must reverse their negative effects by acknowledging their existence and actively trying to make rational decisions instead. Raising awareness of the existence of the unconscious bias leads to a two-pronged benefit: on the one hand, people realise that the problem exists, and on the other hand, when it comes to the role of women in STEM, both men and women can correct their negative thoughts and behaviours towards women, whereas women can more easily spot biased treatment if it occurs. It has in fact emerged from our interviews that women often recognise having been mistreated in hindsight, and as the ‘me too’ movement has proved, raising awareness is a key first step to solving the problem.Importantly, unconscious bias might contribute to the leaky pipeline (i.e. the progressive underrepresentation of women in senior positions) beyond hiring discrimination by affecting women's publication number or even their selection to receive prestigious awards. A recent study, discussed in this news article (Watson, 2021) has uncovered that women's share of international prizes rewarding research excellence lags behind the proportion of professorial positions held by women. The authors identified 141 highly prestigious international prizes – including the Nobel prizes, the Fields Medal for mathematics and the Robert Koch Award for biomedical sciences – and found that the distribution between male and female awardees could not be more different: while these research recognitions were given to 2011 men, they had only been awarded to 262 women between 2001 and 2020. These results do not correlate with lack of quality or quantity of women's research. Instead, implicit bias from award committees, coupled with a lack of proactive efforts to address inequalities in science, reinforces a system that undervalues women. Such studies are of enormous significance, as they help to increase our perception of the systemic factors making women less visible to both the scientific community and society.Unconscious bias is just one of the many issues that, to this day, fuel the leaky pipeline. In addition, common misconceptions about why women get progressively lost along their professional path (i.e. the lack of realistic data concerning women, a widespread topic of concern across numerous fields referred to as the ‘gender data gap’) may lead to the implementation of suboptimal corrective measures. These end up creating a system that is in principle aimed at achieving gender equity, but practically still fails to do so.A clear example of this comes from a survey conducted by Harvard Business School of more than 25,000 graduates, analysing the career trajectories of men and women after completion of their studies (https://hbr.org/2014/12/rethink-what-you-know-about-high-achieving-women). The results, applicable to managerial positions in any field, found that against common belief, women do not ‘opt out’ from their profession in favour of child care, although they do feel the stereotypical societal pressure to do so. After child birth, women rather tend to find themselves in unfulfilling roles with dim prospects for advancement. Thus, despite making more efforts in creating flexible and/or part-time positions for mothers, employers seem to have largely failed to incorporate the value of the intellectual challenges of these roles.The frequent misbelief that part-time working women aim for less challenging work causes women to rate these jobs as not fulfilling, which directly translates to decreased professional satisfaction and a higher probability of discontinuing their careers in such work environments. Furthermore, being part-time automatically takes women off the structured review and promotion ladder in a number of companies, thus introducing a glass ceiling simply based on assumptions, further contributing to the unconscious bias. Individuals, companies and society must re-think, re-evaluate and adjust their inherent beliefs and expectations of women's aspirations in order to sustainably create flexible and inclusive career options for all members of society. This statement does not just pertain to men, it also requires women to become aware of how they might be jeopardized by their own implicit biases and stereotype convictions.Another example of a ‘double-edged sword’ measure, set out to help fix the leaky pipeline but perhaps not completely helpful in reality, relates to how the inclusion of parental leave is differently considered for men and women by funding agencies and employers. First of all, the recognition that pregnancy and birth take a large toll on a woman's body is a step in the right direction, certainly as biology dictates that men cannot take on these roles. In addition to the energy demands and hormonal fluctuations, a woman working in a life science lab is by law excluded from performing many experiments during pregnancy in order to protect the foetus, meaning that she potentially falls behind her colleagues even before birth. Thus, parental leave related to the biological role of pregnancy and birth is absolutely necessary. On the other hand, postnatal care can be shared, but as pointed out during our interviews of professors, current measures don't seem to adequately take fathers into consideration. As an example, mothers are given a greater extension (18 months per child, plus legally documented maternal leave beyond this time) in eligibility than fathers are (duration of paternity leave only) for European Research Grants, the largest funding agency in Europe, a pattern that is similarly followed by other European funding agencies.Notably, government-supported parental leave varies widely among EU countries, meaning that men from countries with little parental leave might not have the ability to take it at all. The important question stemming from such policies is whether they might decrease the incentive or ability for men to take parental leave, especially in couples formed by academics where both careers depend on grant eligibility. If so, the childcare burden can end up still falling predominantly on women. Furthermore, proof of parental leave for fathers is tied to each country's policy, so that any additional time taken may not be considered for grant eligibility and, potentially, on CVs in general. From this it seems clear that, to achieve gender equality, men have to be put in the equation because only the concerted action of men and women can lead to equality. In fact, for women to be granted equal career opportunities in STEM, men have to be motivated and (legally) able to share the unpaid parental duties traditionally performed by women, at least when they are biologically capable of doing so. Furthermore, a revolution of ‘maternity leave’ towards a clear and separate recognition of ‘pregnancy’ and ‘childcare leave’ can ensure fair treatment of child bearers and carers, irrespective of their gender (with positive implications for families not composed of a male-and-female couple) and relieve mothers from their traditionally imposed role as primary caregivers.The fair distribution of parental care duties is a particularly important goal towards equality because childcare is one of the activities contributing to so-called ‘unpaid work’; this includes the obvious household duties and childcare, but also increasingly includes elderly care, all of which to this day still take a higher toll on women than men, even in high-income, developed countries (https://www.oecd.org/dev/development-gender/Unpaid_care_work.pdf). This inequity can negatively impact women's output in their paid jobs, as, for example, became particularly evident during the first COVID-19 lockdown, when women submitted fewer papers to preprint servers and registered fewer new projects compared with men (Viglione, 2020). During one of the ‘STEM fatale Women's Round Table’ discussions at ISTA, a mother/scientist said that without her supportive and equitable relationship, she would never have been able to take on her very demanding high-level job that requires a lot of travel. We think men and women can thus help the situation by making sure that both partners know what's going on regarding domestic duties, with women asking their partners to do more when and if necessary.We would like to conclude this essay with an important piece of advice for young women who want to pursue a career in STEM, coming from our interviewed professors: feel free to follow your goals, do NOT care about what others (peers, as well as society at large) might think, do NOT compromise your goals; STEM is NOT too hard for girls and even if few girls are in STEM at the moment, so what? Being unique is a value!At the end of the day, although representation is tremendously important, young girls should not get discouraged – not having many role models to follow also means they now have the chance of becoming the next!The authors want to thank Professors Carrie Bernecky, Tom Henzinger, Martin Loose and Gaia Novarino for accepting to be interviewed, thus giving significant contribution to the discussion that lead to this article.The authors Nicole Amberg (nicole.amberg@ist.ac.at; Twitter: @amberg_nicole), Melissa Stouffer (melissa.stouffer@ist.ac.at) and Irene Vercellino (irene.vercellino@ist.ac.at; Twitter: @irene50387182) work as a postdoctoral fellows, at the Institute of Science and Technology Austria, Klosterneuburg, Austria.

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.

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.

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).

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.

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.

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).

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.