SUMMARY The generation of faithful cell-type diversity and correct projection neuron numbers is essential for cerebral cortex development. Corticogenesis is however susceptible to genetic interference of critical signaling pathways, including mutations in Mtor/Rptor that lead to microcephaly. How the loss of Rptor/mTORC1 function affects cortical developmental programs, at single cell level, is still unknown. Here, we utilized Mosaic Analysis with Double Markers (MADM) technology to probe Rptor gene function upon sparse single cell- or global tissue-wide ablation. We found that tissue-wide effects drive the etiology of cortical microcephaly upon loss of Rptor, rather than deficits in projection neuron genesis. Conversely, Rptor function is cell-autonomously required for postnatal projection neuron survival in a highly cell-type-specific manner. Collectively, our results suggest that the fine balance of precise cell-type-specific cell-autonomous Rptor/mTORC1 function in concert with non-cell-autonomous tissue-wide effects is essential for the development of a properly-sized cerebral cortex with accurate projection neuron diversity.

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.

Radial glial progenitors (RGPs) generate all projection neurons (PNs) in the cerebral cortex through incompletely understood processes. We combined Mosaic Analysis with Double Markers at embryonic stages (E)12.5 and E13.5 with early postnatal callosal tracing to dissect RGP lineage progression. We find that multipotent RGPs generate all extra-telencephalic (ET) and intra-telencephalic (IT) PNs via parallel sublineages that emerge simultaneously at neurogenesis onset. ET-PN production progresses exclusively via small, self-consuming lineages; IT-PN lineages feature RGPs generating large translaminar outputs. The early emergence of IT-PN-fated RGPs, coinciding with a switch to direct neurogenesis, contributes to the stereotyped population-level progression of the multipotent lineage. We also identify POU3F transcription factors as candidate regulators of IT-PN fate via noncanonical mitotic chromatin binding. The results support a model whereby IT- and ET-PNs arise from an early bifurcation and parallel specification within the multipotent RGP lineage.

Malignant glioma is incurable. Using a mouse genetic mosaic system to generate sporadic Trp53,Nf1-null OPCs, we previously identified oligodendrocyte precursor cell (OPC) as a cell-of-origin of glioma. Here, we report that pre-malignant Trp53,Nf1-null OPCs outcompete wildtype counterparts during their expansion. Blocking competition by mutating/strengthening wildtype OPCs impeded both pre-malignant progression and malignant expansion of glioma. "In-tissue" phosphoproteomic profiling revealed an enrichment of phosphopeptides related to RNA splicing and protein translation at the peak of cell competition, suggesting that competitiveness may stem from unique protein species. Among candidates was mTORC1, whose pharmacological inhibition or genetic disruption resulted in a loss of competitiveness in our mouse model. Finally, analysis of patient biopsies and interrogating the role of individual gliomagenic mutations in OPC competition supported its relevance in human gliomas. Together, these findings identified the driving role of competitive interactions among OPCs in gliomagenesis, and suggest unconventional therapeutic strategies to target this process.

Gene duplication underlies evolutionary innovation, yet many paralogues remain highly similar, raising questions about their functional divergence and physiological relevance. The spliceosomal Sm core protein SNRPB and its mammalian-specific paralogue SNRPN share over 90% sequence identity, but their distinct expression patterns - SNRPB being ubiquitous and SNRPN confined to the brain - suggest specialized functions. Why mammals have two different spliceosomes has remained obscure. Here, we generated isogenic human cell lines expressing ectopically either SNRPB or SNRPN exclusively and found that SNRPN stabilizes transcripts involved in energy metabolism and mitochondrial function, leading to increased mitochondrial abundance and oxygen consumption. Despite similar spliceosomal interactomes, SNRPN more strongly associates with the PRMT5 methylosome complex and exhibits dynamic arginine methylation in its C-terminal region that is sensitive to translation inhibition and amino acid availability. The SNRPN-dependent transcriptome responds to translation inhibition by stabilizing long, intron-rich genes involved in amino acid and energy metabolism. Our findings reveal a nutrient-sensitive, methylation-dependent mechanism that differentiates the two paralogues. This suggests that SNRPN functions as a metabolic-specialized spliceosomal subunit thereby providing tissue-specific adaptation of RNA processing in mammals. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, Individual Grant Project no. 513744403, Scientific Network Grant Project no. 531902894, GRK2526 Genevo - Project no. 407023052, GRK2859 (4R) - Project no. 491145305, Equipment: Project #497669232, Project #524805621, Project #210253511 Ministerium für Bildung, Wissenschaft, Weiterbildung und Kultur, Forschungsinitiative Rheinland-Pfalz (ReALity), Science of Healthy Ageing Research Programme (SHARP) EMBO Young Investigator Program, 5795 Kantone Basel-Stadt & Basel-Landschaft, CH University Medical Center of the Johannes Gutenberg University Mainz, https://ror.org/00q1fsf04, High Potentials Grant program Stiftungen zugunsten der Medizinischen Fakultät der LMU Klinikum, 26069

Radial glial progenitors (RGPs) produce all excitatory neurons in the developing cerebral cortex. Mosaic analysis with double markers (MADM)-based lineage tracing in vivo has revealed a quantitative framework of RGP lineage progression1. Here we established MADM technology2,3 in mouse embryonic stem cells to probe RGP lineage progression in a self-organizing cortical organoid system. We found that RGPs exhibit a high level of plasticity in proliferative potential in organoids rather than strict temporally stereotyped lineage progression as observed in vivo. RGPs in organoids showed increased lineage restriction, diminishing cell-type diversity in clones of cortical projection neurons, despite uniform single-cell transcriptional signatures of RGPs and a unitary lineage trajectory. Thus, critical non-cell-autonomous cues that are absent in self-organizing systems and/or the genuine stem cell niche are essential for faithful temporal control of RGP lineage progression and the generation of clonal cortical cell-type diversity.

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.

The brain vasculature supplies neurons with glucose and oxygen, but little is known about how vascular plasticity contributes to brain function. Using longitudinal in vivo imaging, we report that a substantial proportion of blood vessels in the adult mouse brain sporadically occlude and regress. Their regression proceeds through sequential stages of blood-flow occlusion, endothelial cell collapse, relocation or loss of pericytes, and retraction of glial endfeet. Regressing vessels are found to be widespread in mouse, monkey and human brains. We further reveal that blood vessel regression cause a reduction of neuronal activity due to a dysfunction in mitochondrial metabolism and glutamate production. Our results elucidate the mechanism of vessel regression and its role in neuronal function in the adult brain.

Mosaic Analysis with Double Markers (MADM) represents a mouse genetic approach coupling differential fluorescent labeling to genetic manipulations in dividing cells and their lineages. MADM uniquely enables the generation and visualization of individual control or homozygous mutant cells in a heterozygous genetic environment. Among its diverse applications, MADM has been used to dissect cell-autonomous gene functions important for cortical development and neural development in general. The high cellular resolution offered by MADM also permits the analysis of transcriptomic changes of individual cells upon genetic manipulations. In this chapter, we describe an experimental protocol combining the generation and isolation of MADM-labeled cells with downstream single-cell RNA-sequencing technologies to probe cell-type specific phenotypes due to genetic mutations at single-cell resolution.

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. ### Competing Interest Statement The authors have declared no competing interest. spanish research agency, BES-2016-077737, SEV-2017-0723-18-1, IJC2020-044653-I, JAEICU23EX_0071, PID2021-125618NB-I00, CEX2021-001165-S La Caixa Foundation, E-03-2016-0557140, LCF/BQ/DR21/11880012, LCF/BQ/DI22/11940006, LCF/BQ/PR23/11980051 FWF Austrian Science Fund, https://ror.org/013tf3c58, SFB F78 European Research Council, https://ror.org/0472cxd90, 101118729

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.

In the developing vertebrate central nervous system, neurons and glia typically arise sequentially from common progenitors. Here, we report that the transcription factor Forkhead Box G1 ( Foxg1 ) regulates gliogenesis in the mouse neocortex via distinct cell-autonomous roles in progenitors and postmitotic neurons that regulate different aspects of the gliogenic FGF signalling pathway. We demonstrate that loss of Foxg1 in cortical progenitors at neurogenic stages causes premature astrogliogenesis. We identify a novel FOXG1 target, the pro-gliogenic FGF pathway component Fgfr3 , which is suppressed by FOXG1 cell-autonomously to maintain neurogenesis. Furthermore, FOXG1 can also suppress premature astrogliogenesis triggered by the augmentation of FGF signalling. We identify a second novel function of FOXG1 in regulating the expression of gliogenic cues in newborn neocortical upper-layer neurons. Loss of FOXG1 in postmitotic neurons non-autonomously enhances gliogenesis in the progenitors via FGF signalling. These results fit well with the model that newborn neurons secrete cues that trigger progenitors to produce the next wave of cell types, astrocytes. If FGF signalling is attenuated in Foxg1 null progenitors, they progress to oligodendrocyte production. Therefore, loss of FOXG1 transitions the progenitor to a gliogenic state, producing either astrocytes or oligodendrocytes depending on FGF signalling levels. Our results uncover how FOXG1 integrates extrinsic signalling via the FGF pathway to regulate the sequential generation of neurons, astrocytes, and oligodendrocytes in the cerebral cortex.

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

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

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.

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

Mosaic analysis with double markers (MADM) technology enables the sparse labeling of genetically defined neurons. We present a protocol for time-lapse imaging of cortical projection neuron migration in mice using MADM. We describe steps for the isolation, culturing, and 4D imaging of neuronal dynamics in MADM-labeled brain tissue. While this protocol is compatible with other single-cell labeling methods, the MADM approach provides a genetic platform for the functional assessment of cell-autonomous candidate gene function and the relative contribution of non-cell-autonomous effects. For complete details on the use and execution of this protocol, please refer to Hansen et al. (2022),1 Contreras et al. (2021),2 and Amberg and Hippenmeyer (2021).3

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.