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Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear 1 . The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus 2 and binding diverse proteins 3 – 5 to achieve X-chromosome inactivation (XCI) 6 , 7 . The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off 8 . Here we show that the Xist RNA-binding proteins PTBP1 9 , MATR3 10 , TDP-43 11 and CELF1 12 assemble on the multivalent E-repeat element of Xist 7 and, via self-aggregation and heterotypic protein–protein interactions, form a condensate 1 in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist . Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist- independent XCI phase 8 , indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist -dependence to Xist -independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing. A protein condensate formed by multivalent interactions between the long non-coding RNA Xist and specific RNA-binding proteins drives the compartmentalization required to perpetuate gene silencing on the inactive X chromosome.

The cerebral cortex is a cellularly complex structure comprising a rich diversity of neuronal and glial cell types. Cortical neurons can be broadly categorized into two classes—excitatory neurons that use the neurotransmitter glutamate, and inhibitory interneurons that use γ-aminobutyric acid (GABA). Previous developmental studies in rodents have led to a prevailing model in which excitatory neurons are born from progenitors located in the cortex, whereas cortical interneurons are born from a separate population of progenitors located outside the developing cortex in the ganglionic eminences 1 – 5 . However, the developmental potential of human cortical progenitors has not been thoroughly explored. Here we show that, in addition to excitatory neurons and glia, human cortical progenitors are also capable of producing GABAergic neurons with the transcriptional characteristics and morphologies of cortical interneurons. By developing a cellular barcoding tool called ‘single-cell-RNA-sequencing-compatible tracer for identifying clonal relationships’ (STICR), we were able to carry out clonal lineage tracing of 1,912 primary human cortical progenitors from six specimens, and to capture both the transcriptional identities and the clonal relationships of their progeny. A subpopulation of cortically born GABAergic neurons was transcriptionally similar to cortical interneurons born from the caudal ganglionic eminence, and these cells were frequently related to excitatory neurons and glia. Our results show that individual human cortical progenitors can generate both excitatory neurons and cortical interneurons, providing a new framework for understanding the origins of neuronal diversity in the human cortex. Molecular barcoding is used to show that progenitor cells in the human cortex can produce both excitatory neurons and inhibitory interneurons, with implications for our understanding of the evolution of the human brain.

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.

Cortical function critically depends on inhibitory/excitatory balance. Cortical inhibitory interneurons (cINs) are born in the ventral forebrain and migrate into cortex, where their numbers are adjusted by programmed cell death. Here, we show that loss of clustered gamma protocadherins (Pcdhg), but not of genes in the alpha or beta clusters, increased dramatically cIN BAX-dependent cell death in mice. Surprisingly, electrophysiological and morphological properties of Pcdhg-deficient and wild-type cINs during the period of cIN cell death were indistinguishable. Co-transplantation of wild-type with Pcdhg-deficient interneuron precursors further reduced mutant cIN survival, but the proportion of mutant and wild-type cells undergoing cell death was not affected by their density. Transplantation also allowed us to test for the contribution of Pcdhg isoforms to the regulation of cIN cell death. We conclude that Pcdhg, specifically Pcdhgc3, Pcdhgc4, and Pcdhgc5, play a critical role in regulating cIN survival during the endogenous period of programmed cIN death.

Cortical organoids are self-organizing three-dimensional cultures that model features of the developing human cerebral cortex 1 , 2 . However, the fidelity of organoid models remains unclear 3 – 5 . Here we analyse the transcriptomes of individual primary human cortical cells from different developmental periods and cortical areas. We find that cortical development is characterized by progenitor maturation trajectories, the emergence of diverse cell subtypes and areal specification of newborn neurons. By contrast, organoids contain broad cell classes, but do not recapitulate distinct cellular subtype identities and appropriate progenitor maturation. Although the molecular signatures of cortical areas emerge in organoid neurons, they are not spatially segregated. Organoids also ectopically activate cellular stress pathways, which impairs cell-type specification. However, organoid stress and subtype defects are alleviated by transplantation into the mouse cortex. Together, these datasets and analytical tools provide a framework for evaluating and improving the accuracy of cortical organoids as models of human brain development. Single-cell RNA sequencing clarifies the development and specification of neurons in the human cortex and shows that cell stress impairs this process in cortical organoids.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Highly organized circuits of connected neurons enable diverse brain functions. Improper development of these circuits is associated with neurodevelopmental disorders, and understanding how circuits are formed is crucial for unraveling the mechanisms of these diseases. We currently have an incomplete picture of how specific brain circuits develop and how they are affected in disease, because we lack methods to study them at scale and with single-cell resolution. Monosynaptic rabies tracing is the gold standard method to study circuit architecture. However, it suffers from cellular toxicity, low throughput, lack of control over the timing of labeling, and the inability to access the molecular profiles of individual neurons. To address these issues, we developed an inducible barcoded rabies virus (ibRV) to enable temporal-controlled labeling of synaptic circuits followed by high-throughput single-cell genomics readout. ibRV allows for dissecting neuronal circuit changes over time at single-cell and spatial resolution. We applied ibRV to study the development of specific mouse cortical circuits during late prenatal and postnatal life using single-cell genomics and unbiased spatial transcriptomics as readouts. We characterized and quantified developmental connectivity patterns and molecular cascades that underlie their formation. Additionally, we constructed functional circuit models that enable interrogation of circuit function and dysfunction at specific developmental stages. Our study provides novel tools for circuit analysis and can provide new insights into the mechanisms of mammalian brain development.

In the field of neurodevelopmental biology, what regulates the production of cells and how the final number of cells is determined in the central nervous system (CNS) remains one of the long-standing questions. Indeed, what ultimately determines the brain size and how the final number of neurons is computed in the CNS remains unknown. What makes this question more intriguing is that in the CNS, a supernumerary number of neurons is produced in development. Excess neurons are eliminated postnatally through PCD (PCD). Hence PCD is a developmental feature necessary to establish the final number of neurons in each region of the brain. Yet, how PCD is regulated in the CNS and what molecules are involved in its regulation remains controversial and largely unresolved.In my thesis, I introduce a set of fifty-eight cell-adhesion molecules named clustered protocadherins (Pcdhs) and show evidence that these molecules are key to establishing the final inhibitory neuronal population in the cerebral cortex through the regulation of PCD. The 58 Pcdh genes are tamdebly arranged into three smaller gene clusters: alpha (Pcdhα), beta (Pcdhβ) and gamma (Pcdhγ). Given the genomic complexity of Pcdhs, I first use a series of whole cluster genetic deletions of the Pcdhs gene locus to probe the function of Pcdhα, Pcdhβ or Pcdhγ in the regulation of cIN cell death in mice. Using these cluster deletion mice, we show that Pcdhγ genes, but not Pcdhα or Pcdhβ genes, are required for the survival of approximately 50% of cINs through a BAX-dependent mechanism. I then probe whether cINs compete for survival using PCHDγs by employing a co-transplantation assay I developed. My data suggest that indeed Pcdhγ-deficient and wild-type (WT) cINs of the same age compete for survival in a mechanism that involves Pcdhγ. Surprisingly, three-dimensional reconstructions and patch-clamp recordings indicate that the Pcdhγ mutant cells have similar morphology, excitability and receive similar numbers of inhibitory and excitatory synaptic inputs compared to wild type cINs.Next, I investigated which Pcdhγ genes are key to the regulation of cIN survival. The Pcdhγ gene cluster encodes 22 unique isoforms, which are subclassified as A-type, B-type or C-type isoforms. Importantly, deletion of the C-type isoforms (Pcdhγc3, Pcdhγc4 and Pcdhγc5), but not of the A-type or B-type Pcdhγs, results in neonatal lethality. Hence, to compare in the same host microenvironment the survival of cINs carrying WT or mutant Pcdhγ and to bypass neonatal lethality, I developed a co-transplantation assay using two different reporter systems (GFP and RFP). Using this assay and a series of constitutive Pcdhγ isoform deletions mice, I found that the A- and B-type Pcdhγ have no significant role in cIN PCD. However, the removal of Pcdhγ C-type isoforms (Pcdhγc3, Pcdhγc4 and Pcdhγc5), and in particular the sole removal of Pcdhγc4, resulted in increased cIN cell death. Together these transplantation experiments suggested that survival of cINs largely depends on the expression of Pcdhγc4, but not expression of Pcdhγc3 or Pcdhγc5, in cINs.To complement observations above, I developed lentiviral constructs to overexpress Pcdhγc4 or Pcdhγc5 in cIN precursors that lack the entire Pcdhγ gene set. I found that the expression of Pcdhγc4, but not that of Pcdhγc5, significantly rescued a fraction of cINs destined to die.Lastly and in collaboration with Andrea Hasentaub and Michael Striker laboratories, we probed the activity of cINs carrying or lacking a full set of Pcdhγ genes using two-photon calcium imaging across the period of cell death (data not included).In summary, my thesis work identifies for the first time a cell-adhesion molecule involved in the regulation of PCD in cINs and show evidence that among all Pcdh genes, Pcdhγc4 is key for determining the final number of cINs for the cortex.

Cortical organoids are self-organizing three-dimensional cultures that model features of the developing human cerebral cortex. However, the fidelity of organoid models remains unclear. Here we analyse the transcriptomes of individual primary human cortical cells from different developmental periods and cortical areas. We find that cortical development is characterized by progenitor maturation trajectories, the emergence of diverse cell subtypes and areal specification of newborn neurons. By contrast, organoids contain broad cell classes, but do not recapitulate distinct cellular subtype identities and appropriate progenitor maturation. Although the molecular signatures of cortical areas emerge in organoid neurons, they are not spatially segregated. Organoids also ectopically activate cellular stress pathways, which impairs cell-type specification. However, organoid stress and subtype defects are alleviated by transplantation into the mouse cortex. Together, these datasets and analytical tools provide a framework for evaluating and improving the accuracy of cortical organoids as models of human brain development.

Cortical function critically depends on inhibitory/excitatory balance. Cortical inhibitory interneurons (cINs) are born in the ventral forebrain and migrate into cortex, where their numbers are adjusted by programmed cell death. Previously, we showed that loss of clustered gamma protocadherins ( ), but not of genes in the alpha or beta clusters, increased dramatically cIN BAX-dependent cell death in mice. Here we show that the sole deletion of the Pcdhγc4 isoform, but not of the other 21 isoforms in the Pcdhγ gene cluster, increased cIN cell death in mice during the normal period of programmed cell death. Viral expression of the isoform rescued transplanted cINs lacking from cell death. We conclude that specifically plays a critical role in regulating the survival of cINs during their normal period of cell death. This demonstrates a novel specificity in the role of isoforms in cortical development.