Seminars

Tom Nowakowski explains lineage barcoding and single-cell sequencing approaches for reconstructing neural stem-cell family trees and early wiring programs in developing mouse and human brains.

Neurodevelopmental disorders (NDDs) impose a global burden, affecting an increasing number of individuals. While some causative genes have been identified, understanding the human-specific mechanisms involved in these disorders remains limited. Traditional gene-driven approaches for modeling brain diseases have failed to capture the diverse and convergent mechanisms at play. Centrosomes and cilia act as intermediaries between environmental and intrinsic signals, regulating cellular behavior. Mutations or dosage variations disrupting their function have been linked to brain formation deficits, highlighting their importance, yet their precise contributions remain largely unknown. Hence, we aim to investigate whether the centrosome/cilia axis is crucial for brain development and serves as a hub for human-specific mechanisms disrupted in NDDs. Towards this direction, we first demonstrated species-specific and cell-type-specific differences in the cilia-genes expression during mouse and human corticogenesis. Then, to dissect their role, we provoked their ectopic overexpression or silencing in the developing mouse cortex or in human brain organoids. Our findings suggest that cilia genes manipulation alters both the numbers and the position of NPCs and neurons in the developing cortex. Interestingly, primary cilium morphology is disrupted, as we find changes in their length, orientation and number that lead to disruption of the apical belt and altered delamination profiles during development. Our results give insight into the role of primary cilia in human cortical development and address fundamental questions regarding the diversity and convergence of gene function in development and disease manifestation. It has the potential to uncover novel pharmacological targets, facilitate personalized medicine, and improve the lives of individuals affected by NDDs through targeted cilia-based therapies.

Astrocytes are multifunctional glial cells, implicated in neurogenesis and synaptogenesis, supporting and fine-tuning neuronal activity and maintaining brain homeostasis by controlling blood-brain barrier permeability. During the last years a number of studies have shown that astrocytes can also be converted into neurons if they force-express neurogenic transcription factors or miRNAs. Direct astrocytic reprogramming to induced-neurons (iNs) is a powerful approach for manipulating cell fate, as it takes advantage of the intrinsic neural stem cell (NSC) potential of brain resident reactive astrocytes. To this end, astrocytic cell fate conversion to iNs has been well-established in vitro and in vivo using combinations of transcription factors (TFs) or chemical cocktails. Challenging the expression of lineage-specific TFs is accompanied by changes in the expression of miRNAs, that post-transcriptionally modulate high numbers of neurogenesis-promoting factors and have therefore been introduced, supplementary or alternatively to TFs, to instruct direct neuronal reprogramming. The neurogenic miRNA miR-124 has been employed in direct reprogramming protocols supplementary to neurogenic TFs and other miRNAs to enhance direct neurogenic conversion by suppressing multiple non-neuronal targets. In our group we aimed to investigate whether miR-124 is sufficient to drive direct reprogramming of astrocytes to induced-neurons (iNs) on its own both in vitro and in vivo and elucidate its independent mechanism of reprogramming action. Our in vitro data indicate that miR-124 is a potent driver of the reprogramming switch of astrocytes towards an immature neuronal fate. Elucidation of the molecular pathways being triggered by miR-124 by RNA-seq analysis revealed that miR-124 is sufficient to instruct reprogramming of cortical astrocytes to immature induced-neurons (iNs) in vitro by down-regulating genes with important regulatory roles in astrocytic function. Among these, the RNA binding protein Zfp36l1, implicated in ARE-mediated mRNA decay, was found to be a direct target of miR-124, that be its turn targets neuronal-specific proteins participating in cortical development, which get de-repressed in miR-124-iNs. Furthermore, miR-124 is potent to guide direct neuronal reprogramming of reactive astrocytes to iNs of cortical identity following cortical trauma, a novel finding confirming its robust reprogramming action within the cortical microenvironment under neuroinflammatory conditions. In parallel to their reprogramming properties, astrocytes also participate in the maintenance of blood-brain barrier integrity, which ensures the physiological functioning of the central nervous system and gets affected contributing to the pathology of several neurodegenerative diseases. To study in real time the dynamic physical interactions of astrocytes with brain vasculature under homeostatic and pathological conditions, we performed 2-photon brain intravital imaging in a mouse model of systemic neuroinflammation, known to trigger astrogliosis and microgliosis and to evoke changes in astrocytic contact with brain vasculature. Our in vivo findings indicate that following neuroinflammation the endfeet of activated perivascular astrocytes lose their close proximity and physiological cross-talk with vasculature, however this event is at compensated by the cross-talk of astrocytes with activated microglia, safeguarding blood vessel coverage and maintenance of blood-brain integrity.

Cellular crosstalk is an essential process during brain development and it is influenced by numerous factors, including the morphology of the cells, their adhesion molecules, the local extracellular matrix and the secreted vesicles. Inspired by mutations associated with neurodevelopmental disorders, we focus on understanding the role of extracellular mechanisms essential for the correct development of the human brain. Hence, we combine the in vivo mouse model and the in vitro human-derived neurons, cerebral organoids, and dorso-ventral assembloids in order to better comprehend the molecular and cellular mechanisms involved in ventral progenitors’ proliferation and fate as well as migration and maturation of inhibitory neurons during human brain development and tackle the causes of neurodevelopmental disorders. We particularly focus on mutations in genes influencing cell-cell contacts, extracellular matrix, and secretion of vesicles and therefore study intrinsic and extrinsic mechanisms contributing to the formation of the brain. Our data reveal an important contribution of cell non-autonomous mechanisms in the development of neurodevelopmental disorders.

The cerebral cortex contains an extraordinary diversity of excitatory projection neuron (PN) and inhibitory interneurons (IN), wired together to form complex circuits. Spatiotemporally coordinated execution of intrinsic molecular programs by PNs and INs and activity-dependent processes, contribute to cortical development and cortical microcircuits formation. Alterations of these delicate processes have often been associated to neurological/neurodevelopmental disorders. However, despite the groundbreaking discovery that spontaneous activity in the embryonic brain can shape regional identities of distinct cortical territories, it is still unclear whether this early activity contributes to define subtype-specific neuronal fate as well as circuit assembly. In this study, we combined in utero genetic perturbations via CRISPR/Cas9 system and pharmacological inhibition of selected ion channels with RNA-sequencing and live imaging technologies to identify the activity-regulated processes controlling the development of different cortical PN classes, their wiring and the acquisition of subtype specific features. Moreover, we generated human induced pluripotent stem cells (iPSCs) form patients affected by a severe, rare and untreatable form of developmental epileptic encephalopathy. By differentiating cortical organoids form patient-derived iPSCs we create human models of early electrical alterations for studying molecular, structural and functional consequences of the genetic mutations during cortical development. Our ultimate goal is to define the activity-conditioned processes that physiologically occur during the development of cortical circuits, to identify novel therapeutical paths to address the pathological consequences of neonatal epilepsies.

The development of human induced pluripotent stem cells (iPSC) and their subsequent differentiation into neurons has provided new opportunities for the generation of physiologically-relevant, in vitro disease models. I will present our work using iPSC to modal familial Alzheimer's Disease (fAD) and Frontotemporal Dementia (FTD). We have investigated the mutation-specific effects of APP and PSEN1 mutations on Abeta generation in neurons generated from individuals with fAD, revealing distinct mechanisms that may contribute to clinical heterogeneity in disease. I will also discuss our work to understand the developmental and pathological changes to tau that occur in iPSC-neurons, particularly the challenges of understanding tau pathology in a developmental system, tau proteostasis and how iPSC-neurons may help us identify early signatures of tau pathology in disease.

Three intimately related dimensions of the mammalian genome—linear DNA sequence, gene transcription, and 3D genome architecture—are crucial for the development of nervous systems. Changes in the linear genome (e.g., de novo mutations), transcriptome, and 3D genome structure lead to debilitating neurodevelopmental disorders, such as autism and schizophrenia. However, current technologies and data are severely limited: (1) 3D genome structures of single brain cells have not been solved; (2) little is known about the dynamics of single-cell transcriptome and 3D genome after birth; (3) true de novo mutations are extremely difficult to distinguish from false positives (DNA damage and/or amplification errors). Here, I filled in this longstanding technological and knowledge gap. I recently developed a high-resolution method—diploid chromatin conformation capture (Dip-C)—which resolved the first 3D structure of the human genome, tackling a longstanding problem dating back to the 1880s. Using Dip-C, I obtained the first 3D genome structure of a single brain cell, and created the first transcriptome and 3D genome atlas of the mouse brain during postnatal development. I found that in adults, 3D genome “structure types” delineate all major cell types, with high correlation between chromatin A/B compartments and gene expression. During development, both transcriptome and 3D genome are extensively transformed in the first month of life. In neurons, 3D genome is rewired across scales, correlated with gene expression modules, and independent of sensory experience. Finally, I examined allele-specific structure of imprinted genes, revealing local and chromosome-wide differences. More recently, I expanded my 3D genome atlas to the human and mouse cerebellum—the most consistently affected brain region in autism. I uncovered unique 3D genome rewiring throughout life, providing a structural basis for the cerebellum’s unique mode of development and aging. In addition, to accurately measure de novo mutations in a single cell, I developed a new method—multiplex end-tagging amplification of complementary strands (META-CS), which eliminates nearly all false positives by virtue of DNA complementarity. Using META-CS, I determined the true mutation spectrum of single human brain cells, free from chemical artifacts. Together, my findings uncovered an unknown dimension of neurodevelopment, and open up opportunities for new treatments for autism and other developmental disorders.

The Hsieh lab focuses on the mechanisms that promote neural stem cell self-renewal and differentiation in embryonic and adult brain. Using mouse models, video-EEG monitoring, viral techniques, and imaging/electrophysiological approaches, we elucidated many of the key transcriptional/epigenetic regulators of adult neurogenesis and showed aberrant new neuron integration in adult rodent hippocampus contribute to circuit disruption and seizure development. Building on this work, I will present our recent studies describing how GABA-mediated Ca2+ activity regulates the production of aberrant adult-born granule cells. In a new direction of my laboratory, we are using human induced pluripotent stem cells and brain organoid models as approaches to understand brain development and disease. Mutations in one gene, Aristaless-related homeobox (ARX), are of considerable interest since they are known to cause a common spectrum of neurodevelopmental disorders including epilepsy, autism, and intellectual disability. We have generated cortical and subpallial organoids from patients with poly-alanine expansion mutations in ARX. To understand the nature of ARX mutations in the organoid system, we are currently performing cellular, molecular, and physiological analyses. I will present these data to gain a comprehensive picture of the effect of ARX mutations in brain development. Since we do not understand how human brain development is affected by ARX mutations that contribute to epilepsy, we believe these studies will allow us to understand the mechanism of pathogenesis of ARX mutations, which has the potential to impact the diagnosis and care of patients.

Neural stem cells (NSCs) generate new neurons throughout life. We use imaging-, genome editing-, and transgenesis-based approaches as well as cellular models of human diseases using pluripotent embryonic cells to study the molecular and cellular framework of NSC biology in the developing and adult brain. Aim of our research is to understand how physiologic and disease-associated alterations of neurogenesis are translated into stem cell-associated plastic changes in the developing and adult brain on a molecular, cellular, and behavioral level.

Recent findings have revealed that mRNAs have a much more dynamic behaviour than initially described. This is particularly true in neurons, where mRNAs are transported to specific axonal and dendritic areas. The seminar will present our most recent findings unveiling complex mRNA processing dynamics driven by splicing proteins in developing axons.

The symposium will start with Prof Song-Hai Shi who will present “Assembly of the neocortex”. Then, Dr Lynette Lim will talk about “Shared and Unique Developmental Trajectories of Cortical Inhibitory Neurons”. Dr Alfredo Molina will deal with the “Tuneable progenitor cells to build the cerebral cortex”, and Prof Tomasz Nowakowski will present “Charting the molecular 'protomap' of the human cerebral cortex using single cell genomic”.

The symposium will start with Prof Cooper who will present “From neural tube to neocortex: the role of adhesion in maintaining stem cell morphology and function”. Then, Dr Tsai will talk about “In the search for new genes involved in brain development and disorders”. Dr Del Pino will deal with the “Regulation of intrinsic network activity during area patterning in the cerebral cortex”, and Dr Wang will present “Modelling Neurodevelopmental Disorders in Flies”.

The establishment of synaptic connections is essential for normal brain function, yet the molecular mechanisms responsible for the precise connectivity of specific neural circuits remain largely unknown. Previous work has shown that the assembly of cortical circuits requires specific functions of molecular signalling complexes at different classes of synapses. In this talk, I will describe the molecular logic through which specific pyramidal cell-interneuron circuits are established in the cerebral cortex of the mouse, and how alterations in some of these connectivity motifs might be liked to disease.

Corticospinal neurons (CSN) are the cortical projection neurons that innervate the spinal cord and some brainstem targets with segmental precision to control voluntary movement of specific functional motor groups, limb sections, or individual digits, yet molecular regulation over CSN segmental target specificity is essentially unknown. CSN subpopulations exhibit striking axon targeting specificity from development into maturity: Evolutionarily newer rostrolateral CSN exclusively innervate bulbar-cervical targets (CSNBC-lat), while evolutionarily older caudomedial CSN (CSNmed) are more heterogeneous, with distinct subpopulations extending axons to either bulbar-cervical or thoraco-lumbar segments. The cervical cord, with its evolutionarily enhanced precision of forelimb movement, is innervated by multiple CSN subpopulations, suggesting inter-neuronal interactions in establishing corticospinal connectivity. I identify that Lumican, previously unrecognized in axon development, controls the specificity of cervical spinal cord innervation by CSN. Remarkably, Lumican, an extracellular matrix protein expressed by CSNBC-lat, non-cell-autonomously suppresses axon collateralization in the cervical cord by CSNmed. Intersectional viral labeling and mouse genetics further identify that Lumican controls axon collateralization by multiple subpopulations in caudomedial sensorimotor cortex. These results identify inter-axonal molecular crosstalk between CSN subpopulations as a novel mechanism controlling corticospinal connectivity and competitive specificity. Further, this mechanism has potential implications for evolutionary diversification of corticospinal circuitry with finer scale precision. "" Complementing this work, to comprehensively elucidate related axon projection mechanisms functioning at tips of growing CSN axons in vivo, I am currently applying experimental and analytic approaches recently developed in my postdoc lab (Poulopoulos*, Murphy*, Nature, 2019) to quantitatively and subcellularly “map” RNA and protein molecular machinery of subtype-specific growth cones, in parallel to their parent somata, isolated directly in vivo from developing subcerebral projection neurons (SCPN; the broader cortical output neuron population targeting both brainstem and spinal cord; includes CSN). I am investigating both normal development and GC-soma dysregulation with mutation of central CSN-SCPN transcriptional regulator Ctip2/Bcl11b.