Publications by authors named "Tomasz J Nowakowski"

47 Publications

Picroscope: low-cost system for simultaneous longitudinal biological imaging.

Commun Biol 2021 Nov 4;4(1):1261. Epub 2021 Nov 4.

Department of Electrical and Computer Engineering, University of California Santa Cruz, Santa Cruz, CA, 95060, USA.

Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here, we propose the Picroscope, which is the first low-cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here, we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish, and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long-time periods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-021-02779-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8569150PMC
November 2021

Light-weight electrophysiology hardware and software platform for cloud-based neural recording experiments.

J Neural Eng 2021 Nov 12;18(6). Epub 2021 Nov 12.

Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95060, United States of America.

Neural activity represents a functional readout of neurons that is increasingly important to monitor in a wide range of experiments. Extracellular recordings have emerged as a powerful technique for measuring neural activity because these methods do not lead to the destruction or degradation of the cells being measured. Current approaches to electrophysiology have a low throughput of experiments due to manual supervision and expensive equipment. This bottleneck limits broader inferences that can be achieved with numerous long-term recorded samples.We developed Piphys, an inexpensive open source neurophysiological recording platform that consists of both hardware and software. It is easily accessed and controlled via a standard web interface through Internet of Things (IoT) protocols.We used a Raspberry Pi as the primary processing device along with an Intan bioamplifier. We designed a hardware expansion circuit board and software to enable voltage sampling and user interaction. This standalone system was validated with primary human neurons, showing reliability in collecting neural activity in near real-time.The hardware modules and cloud software allow for remote control of neural recording experiments as well as horizontal scalability, enabling long-term observations of development, organization, and neural activity at scale.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1088/1741-2552/ac310aDOI Listing
November 2021

An atlas of cortical arealization identifies dynamic molecular signatures.

Nature 2021 10 6;598(7879):200-204. Epub 2021 Oct 6.

Department of Neurology, University of California, San Francisco, San Francisco, CA, USA.

The human brain is subdivided into distinct anatomical structures, including the neocortex, which in turn encompasses dozens of distinct specialized cortical areas. Early morphogenetic gradients are known to establish early brain regions and cortical areas, but how early patterns result in finer and more discrete spatial differences remains poorly understood. Here we use single-cell RNA sequencing to profile ten major brain structures and six neocortical areas during peak neurogenesis and early gliogenesis. Within the neocortex, we find that early in the second trimester, a large number of genes are differentially expressed across distinct cortical areas in all cell types, including radial glia, the neural progenitors of the cortex. However, the abundance of areal transcriptomic signatures increases as radial glia differentiate into intermediate progenitor cells and ultimately give rise to excitatory neurons. Using an automated, multiplexed single-molecule fluorescent in situ hybridization approach, we find that laminar gene-expression patterns are highly dynamic across cortical regions. Together, our data suggest that early cortical areal patterning is defined by strong, mutually exclusive frontal and occipital gene-expression signatures, with resulting gradients giving rise to the specification of areas between these two poles throughout successive developmental timepoints.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-021-03910-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8494648PMC
October 2021

Single-cell epigenomics reveals mechanisms of human cortical development.

Nature 2021 10 6;598(7879):205-213. Epub 2021 Oct 6.

Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA.

During mammalian development, differences in chromatin state coincide with cellular differentiation and reflect changes in the gene regulatory landscape. In the developing brain, cell fate specification and topographic identity are important for defining cell identity and confer selective vulnerabilities to neurodevelopmental disorders. Here, to identify cell-type-specific chromatin accessibility patterns in the developing human brain, we used a single-cell assay for transposase accessibility by sequencing (scATAC-seq) in primary tissue samples from the human forebrain. We applied unbiased analyses to identify genomic loci that undergo extensive cell-type- and brain-region-specific changes in accessibility during neurogenesis, and an integrative analysis to predict cell-type-specific candidate regulatory elements. We found that cerebral organoids recapitulate most putative cell-type-specific enhancer accessibility patterns but lack many cell-type-specific open chromatin regions that are found in vivo. Systematic comparison of chromatin accessibility across brain regions revealed unexpected diversity among neural progenitor cells in the cerebral cortex and implicated retinoic acid signalling in the specification of neuronal lineage identity in the prefrontal cortex. Together, our results reveal the important contribution of chromatin state to the emerging patterns of cell type diversity and cell fate specification and provide a blueprint for evaluating the fidelity and robustness of cerebral organoids as a model for cortical development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-021-03209-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8494642PMC
October 2021

Human microglia states are conserved across experimental models and regulate neural stem cell responses in chimeric organoids.

Cell Stem Cell 2021 Sep 16. Epub 2021 Sep 16.

Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA; Department of Psychiatry, University of California, San Francisco, San Francisco, CA, USA; Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA; Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA. Electronic address:

Microglia are resident macrophages in the brain that emerge in early development and respond to the local environment by altering their molecular and phenotypic states. Fundamental questions about microglia diversity and function during development remain unanswered because we lack experimental strategies to interrogate their interactions with other cell types and responses to perturbations ex vivo. We compared human microglia states across culture models, including cultured primary and pluripotent stem cell-derived microglia. We developed a "report card" of gene expression signatures across these distinct models to facilitate characterization of their responses across experimental models, perturbations, and disease conditions. Xenotransplantation of human microglia into cerebral organoids allowed us to characterize key transcriptional programs of developing microglia in vitro and reveal that microglia induce transcriptional changes in neural stem cells and decrease interferon signaling response genes. Microglia additionally accelerate the emergence of synchronized oscillatory network activity in brain organoids by modulating synaptic density.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2021.08.015DOI Listing
September 2021

UCSC Cell Browser: Visualize Your Single-Cell Data.

Bioinformatics 2021 Jul 9. Epub 2021 Jul 9.

Genomics Institute, University of California Santa Cruz, Santa Cruz, CA, USA.

Summary: As the use of single-cell technologies has grown, so has the need for tools to explore these large, complicated datasets. The UCSC Cell Browser is a tool that allows scientists to visualize gene expression and metadata annotation distribution throughout a single-cell dataset or multiple datasets.

Availability And Implementation: We provide the UCSC Cell Browser as a free website where scientists can explore a growing collection of single-cell datasets and a freely available python package for scientists to create stable, self-contained visualizations for their own single-cell datasets. Learn more at https://cells.ucsc.edu.

Supplementary Information: Supplementary data are available at Bioinformatics online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btab503DOI Listing
July 2021

Distinct nuclear compartment-associated genome architecture in the developing mammalian brain.

Nat Neurosci 2021 09 8;24(9):1235-1242. Epub 2021 Jul 8.

Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA.

Nuclear compartments are thought to play a role in three-dimensional genome organization and gene expression. In mammalian brain, the architecture and dynamics of nuclear compartment-associated genome organization is not known. In this study, we developed Genome Organization using CUT and RUN Technology (GO-CaRT) to map genomic interactions with two nuclear compartments-the nuclear lamina and nuclear speckles-from different regions of the developing mouse, macaque and human brain. Lamina-associated domain (LAD) architecture in cells in vivo is distinct from that of cultured cells, including major differences in LADs previously considered to be cell type invariant. In the mouse and human forebrain, dorsal and ventral neural precursor cells have differences in LAD architecture that correspond to their regional identity. LADs in the human and mouse cortex contain transcriptionally highly active sub-domains characterized by broad depletion of histone-3-lysine-9 dimethylation. Evolutionarily conserved LADs in human, macaque and mouse brain are enriched for transcriptionally active neural genes associated with synapse function. By integrating GO-CaRT maps with genome-wide association study data, we found speckle-associated domains to be enriched for schizophrenia risk loci, indicating a physical relationship between these disease-associated genetic variants and a specific nuclear structure. Our work provides a framework for understanding the relationship between distinct nuclear compartments and genome function in brain development and disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41593-021-00879-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410652PMC
September 2021

The CHD8/CHD7/Kismet family links blood-brain barrier glia and serotonin to ASD-associated sleep defects.

Sci Adv 2021 Jun 4;7(23). Epub 2021 Jun 4.

Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud university medical center, 6525 GA, Nijmegen, Netherlands.

Sleep disturbances in autism and neurodevelopmental disorders are common and adversely affect patient's quality of life, yet the underlying mechanisms are understudied. We found that individuals with mutations in , among the highest-confidence autism risk genes, or suffer from disturbed sleep maintenance. These defects are recapitulated in mutants affecting , the sole ortholog. We show that Kismet is required in glia for early developmental and adult sleep architecture. This role localizes to subperineurial glia constituting the blood-brain barrier. We demonstrate that Kismet-related sleep disturbances are caused by high serotonin during development, paralleling a well-established but genetically unsolved autism endophenotype. Despite their developmental origin, Kismet's sleep architecture defects can be reversed in adulthood by a behavioral regime resembling human sleep restriction therapy. Our findings provide fundamental insights into glial regulation of sleep and propose a causal mechanistic link between the CHD8/CHD7/Kismet family, developmental hyperserotonemia, and autism-associated sleep disturbances.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.abe2626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8177706PMC
June 2021

Single-cell atlas of early human brain development highlights heterogeneity of human neuroepithelial cells and early radial glia.

Nat Neurosci 2021 04 15;24(4):584-594. Epub 2021 Mar 15.

Department of Neurology, University of California, San Francisco (UCSF), San Francisco, CA, USA.

The human cortex comprises diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41593-020-00794-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8012207PMC
April 2021

Parallel in vivo analysis of large-effect autism genes implicates cortical neurogenesis and estrogen in risk and resilience.

Neuron 2021 03 25;109(5):788-804.e8. Epub 2021 Jan 25.

Department of Psychiatry and Behavioral Sciences, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94143, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94143, USA; Langley Porter Psychiatric Institute, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Gene Ontology analyses of autism spectrum disorders (ASD) risk genes have repeatedly highlighted synaptic function and transcriptional regulation as key points of convergence. However, these analyses rely on incomplete knowledge of gene function across brain development. Here we leverage Xenopus tropicalis to study in vivo ten genes with the strongest statistical evidence for association with ASD. All genes are expressed in developing telencephalon at time points mapping to human mid-prenatal development, and mutations lead to an increase in the ratio of neural progenitor cells to maturing neurons, supporting previous in silico systems biological findings implicating cortical neurons in ASD vulnerability, but expanding the range of convergent functions to include neurogenesis. Systematic chemical screening identifies that estrogen, via Sonic hedgehog signaling, rescues this convergent phenotype in Xenopus and human models of brain development, suggesting a resilience factor that may mitigate a range of ASD genetic risks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2021.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132462PMC
March 2021

The Expanding Cell Diversity of the Brain Vasculature.

Front Physiol 2020 3;11:600767. Epub 2020 Dec 3.

Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, United States.

The cerebrovasculature is essential to brain health and is tasked with ensuring adequate delivery of oxygen and metabolic precursors to ensure normal neurologic function. This is coordinated through a dynamic, multi-directional cellular interplay between vascular, neuronal, and glial cells. Molecular exchanges across the blood-brain barrier or the close matching of regional blood flow with brain activation are not uniformly assigned to arteries, capillaries, and veins. Evidence has supported functional segmentation of the brain vasculature. This is achieved in part through morphologic or transcriptional heterogeneity of brain vascular cells-including endothelium, pericytes, and vascular smooth muscle. Advances with single cell genomic technologies have shown increasing cell complexity of the brain vasculature identifying previously unknown cell types and further subclassifying transcriptional diversity in cardinal vascular cell types. Cell-type specific molecular transitions or zonations have been identified. In this review, we summarize emerging evidence for the expanding vascular cell diversity in the brain and how this may provide a cellular basis for functional segmentation along the arterial-venous axis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphys.2020.600767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744630PMC
December 2020

Are Organoids Ready for Prime Time?

Cell Stem Cell 2020 09;27(3):361-365

Department of Anatomy, University of California, San Francisco, CA, USA; Department of Psychiatry and Behavioral Sciences, University of California, San Francisco, CA, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA. Electronic address:

Innovations in organoid-based models of human tissues have made them an exciting experimental platform for studying development and disease. However, these models require systematic benchmarking against primary tissue to establish their value. We discuss key parameters that impact the utility of organoid models, primarily focusing on cerebral organoids as examples.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2020.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7559415PMC
September 2020

Revealing architectural order with quantitative label-free imaging and deep learning.

Elife 2020 07 27;9. Epub 2020 Jul 27.

Chan Zuckerberg Biohub, San Francisco, United States.

We report quantitative label-free imaging with phase and polarization (QLIPP) for simultaneous measurement of density, anisotropy, and orientation of structures in unlabeled live cells and tissue slices. We combine QLIPP with deep neural networks to predict fluorescence images of diverse cell and tissue structures. QLIPP images reveal anatomical regions and axon tract orientation in prenatal human brain tissue sections that are not visible using brightfield imaging. We report a variant of U-Net architecture, multi-channel 2.5D U-Net, for computationally efficient prediction of fluorescence images in three dimensions and over large fields of view. Further, we develop data normalization methods for accurate prediction of myelin distribution over large brain regions. We show that experimental defects in labeling the human tissue can be rescued with quantitative label-free imaging and neural network model. We anticipate that the proposed method will enable new studies of architectural order at spatial scales ranging from organelles to tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.55502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431134PMC
July 2020

A Chromatin Accessibility Atlas of the Developing Human Telencephalon.

Cell 2020 08 30;182(3):754-769.e18. Epub 2020 Jun 30.

Department of Psychiatry, Langley Porter Psychiatric Institute, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, USA. Electronic address:

To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415678PMC
August 2020

Cell stress in cortical organoids impairs molecular subtype specification.

Nature 2020 02 29;578(7793):142-148. Epub 2020 Jan 29.

Department of Neurology, University of California, San Francisco (UCSF), San Francisco, CA, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-1962-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7433012PMC
February 2020

Evolutionary Expansion of Human Cerebellar Germinal Zones.

Trends Neurosci 2020 02 15;43(2):75-77. Epub 2020 Jan 15.

Department of Anatomy, University of California, San Francisco, CA, USA; Department of Psychiatry, University of California, San Francisco, CA, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA. Electronic address:

Haldipur et al. explored the developmental origins of the human cerebellum, which has gained growing appreciation for its involvement in human cognition. The authors discovered human-unique expansion and maintenance of cerebellar germinal zones, reminiscent of processes in the developing human cerebral cortex necessary for generating expanded neuronal populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tins.2019.12.005DOI Listing
February 2020

Disruptive mutations in TANC2 define a neurodevelopmental syndrome associated with psychiatric disorders.

Nat Commun 2019 10 15;10(1):4679. Epub 2019 Oct 15.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA.

Postsynaptic density (PSD) proteins have been implicated in the pathophysiology of neurodevelopmental and psychiatric disorders. Here, we present detailed clinical and genetic data for 20 patients with likely gene-disrupting mutations in TANC2-whose protein product interacts with multiple PSD proteins. Pediatric patients with disruptive mutations present with autism, intellectual disability, and delayed language and motor development. In addition to a variable degree of epilepsy and facial dysmorphism, we observe a pattern of more complex psychiatric dysfunction or behavioral problems in adult probands or carrier parents. Although this observation requires replication to establish statistical significance, it also suggests that mutations in this gene are associated with a variety of neuropsychiatric disorders consistent with its postsynaptic function. We find that TANC2 is expressed broadly in the human developing brain, especially in excitatory neurons and glial cells, but shows a more restricted pattern in Drosophila glial cells where its disruption affects behavioral outcomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-12435-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794285PMC
October 2019

Development and Arealization of the Cerebral Cortex.

Neuron 2019 09;103(6):980-1004

The Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research at the University of California, San Francisco, San Francisco, CA 94143, USA; Department of Anatomy, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Adult cortical areas consist of specialized cell types and circuits that support unique higher-order cognitive functions. How this regional diversity develops from an initially uniform neuroepithelium has been the subject of decades of seminal research, and emerging technologies, including single-cell transcriptomics, provide a new perspective on area-specific molecular diversity. Here, we review the early developmental processes that underlie cortical arealization, including both cortex intrinsic and extrinsic mechanisms as embodied by the protomap and protocortex hypotheses, respectively. We propose an integrated model of serial homology whereby intrinsic genetic programs and local factors establish early transcriptomic differences between excitatory neurons destined to give rise to broad "proto-regions," and activity-dependent mechanisms lead to progressive refinement and formation of sharp boundaries between functional areas. Finally, we explore the potential of these basic developmental processes to inform our understanding of the emergence of functional neural networks and circuit abnormalities in neurodevelopmental disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2019.07.009DOI Listing
September 2019

Human brain development through the lens of cerebral organoid models.

Brain Res 2019 12 19;1725:146470. Epub 2019 Sep 19.

Department of Anatomy, University of California, San Francisco, CA, USA; Department of Psychiatry, University of California, San Francisco, CA, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA. Electronic address:

The brain is one of the most complex organs in the body, which emerges from a relatively simple set of basic 'building blocks' during early development according to complex cellular and molecular events orchestrated through a set of inherited instructions. Innovations in stem cell technologies have enabled modelling of neural cells using two- and three-dimensional cultures. In particular, cerebral ('brain') organoids have taken the center stage of brain development models that have the potential for providing meaningful insight into human neurodevelopmental and neurological disorders. We review the current understanding of cellular events during human brain organogenesis, and the events occurring during cerebral organoid differentiation. We highlight the strengths and weaknesses of this experimental model system. In particular, we explain evidence that organoids can mimic many aspects of early neural development, including neural induction, patterning, and broad neurogenesis and gliogenesis programs, offering the opportunity to study genetic regulation of these processes in a human context. Several shortcomings of the current culture methods limit the utility of cerebral organoids to spontaneously give rise to many important cell types, and to model higher order features of tissue organization. We suggest that future studies aim to improve these features in order to make them better models for the study of laminar organization, circuit formation and how disruptions of these processes relate to disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.brainres.2019.146470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887101PMC
December 2019

Automated four-dimensional long term imaging enables single cell tracking within organotypic brain slices to study neurodevelopment and degeneration.

Commun Biol 2019 1;2:155. Epub 2019 May 1.

Gladstone Center for Systems and Therapeutics, San Francisco, CA 94158 USA.

Current approaches for dynamic profiling of single cells rely on dissociated cultures, which lack important biological features existing in tissues. Organotypic slice cultures preserve aspects of structural and synaptic organisation within the brain and are amenable to microscopy, but established techniques are not well adapted for high throughput or longitudinal single cell analysis. Here we developed a custom-built, automated confocal imaging platform, with improved organotypic slice culture and maintenance. The approach enables fully automated image acquisition and four-dimensional tracking of morphological changes within individual cells in organotypic cultures from rodent and human primary tissues for at least 3 weeks. To validate this system, we analysed neurons expressing a disease-associated version of huntingtin (HTT586Q138-EGFP), and observed that they displayed hallmarks of Huntington's disease and died sooner than controls. By facilitating longitudinal single-cell analyses of neuronal physiology, our system bridges scales necessary to attain statistical power to detect developmental and disease phenotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-019-0411-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494885PMC
April 2020

Multimodal Single-Cell Analysis Reveals Physiological Maturation in the Developing Human Neocortex.

Neuron 2019 04 12;102(1):143-158.e7. Epub 2019 Feb 12.

Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types. By combining measurements of intracellular Ca elevations in response to neurotransmitter receptor agonists and RNA sequencing of the same single cells, we show that Ca responses are cell-type-specific and change dynamically with lineage progression. Physiological response properties predict molecular cell identity and additionally reveal diversity not captured by single-cell transcriptomics. We find that the serotonin receptor HTR2A selectively activates radial glia cells in the developing human, but not mouse, neocortex, and inhibiting HTR2A receptors in human radial glia disrupts the radial glial scaffold. We show highly specific neurotransmitter signaling during neurogenesis in the developing human neocortex and highlight evolutionarily divergent mechanisms of physiological signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2019.01.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648658PMC
April 2019

Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution.

Cell 2019 02;176(4):743-756.e17

Department of Neurology, University of California, San Francisco (UCSF), San Francisco, CA, USA; The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, CA, USA. Electronic address:

Direct comparisons of human and non-human primate brains can reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. Despite metabolic differences, organoid models preserve gene regulatory networks related to primary cell types and developmental processes. We further identified 261 differentially expressed genes in human compared to both chimpanzee organoids and macaque cortex, enriched for recent gene duplications, and including multiple regulators of PI3K-AKT-mTOR signaling. We observed increased activation of this pathway in human radial glia, dependent on two receptors upregulated specifically in human: INSR and ITGB8. Our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.01.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544371PMC
February 2019

Mafb and c-Maf Have Prenatal Compensatory and Postnatal Antagonistic Roles in Cortical Interneuron Fate and Function.

Cell Rep 2019 01;26(5):1157-1173.e5

Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94158, USA; Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

Mafb and c-Maf transcription factor (TF) expression is enriched in medial ganglionic eminence (MGE) lineages, beginning in late-secondary progenitors and continuing into mature parvalbumin (PV) and somatostatin (SST) interneurons. However, the functions of Maf TFs in MGE development remain to be elucidated. Herein, Mafb and c-Maf were conditionally deleted, alone and together, in the MGE and its lineages. Analyses of Maf mutant mice revealed redundant functions of Mafb and c-Maf in secondary MGE progenitors, where they repress the generation of SST cortical and hippocampal interneurons. By contrast, Mafb and c-Maf have distinct roles in postnatal cortical interneuron (CIN) morphological maturation, synaptogenesis, and cortical circuit integration. Thus, Mafb and c-Maf have redundant and opposing functions at different steps in CIN development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.01.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602795PMC
January 2019

Neuroserpin expression during human brain development and in adult brain revealed by immunohistochemistry and single cell RNA sequencing.

J Anat 2019 09 15;235(3):543-554. Epub 2019 Jan 15.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.

Neuroserpin is a serine-protease inhibitor mainly expressed in the CNS and involved in the inhibition of the proteolytic cascade. Animal models confirmed its neuroprotective role in perinatal hypoxia-ischaemia and adult stroke. Although neuroserpin may be a potential therapeutic target in the treatment of the aforementioned conditions, there is still no information in the literature on its distribution during human brain development. The present study provides a detailed description of the changing spatiotemporal patterns of neuroserpin focusing on physiological human brain development. Five stages were distinguished within our examined age range which spanned from the 7th gestational week until adulthood. In particular, subplate and deep cortical plate neurons were identified as the main sources of neuroserpin production between the 25th gestational week and the first postnatal month. Our immunohistochemical findings were substantiated by single cell RNA sequencing data showing specific neuronal and glial cell types expressing neuroserpin. The characterization of neuroserpin expression during physiological human brain development is essential for forthcoming studies which will explore its involvement in pathological conditions, such as perinatal hypoxia-ischaemia and adult stroke in human.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/joa.12931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704272PMC
September 2019

Regulation of cell-type-specific transcriptomes by microRNA networks during human brain development.

Nat Neurosci 2018 12 19;21(12):1784-1792. Epub 2018 Nov 19.

Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA, USA.

MicroRNAs (miRNAs) regulate many cellular events during brain development by interacting with hundreds of mRNA transcripts. However, miRNAs operate nonuniformly upon the transcriptional profile with an as yet unknown logic. Shortcomings in defining miRNA-mRNA networks include limited knowledge of in vivo miRNA targets and their abundance in single cells. By combining multiple complementary approaches, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation with an antibody to AGO2 (AGO2-HITS-CLIP), single-cell profiling and computational analyses using bipartite and coexpression networks, we show that miRNA-mRNA interactions operate as functional modules that often correspond to cell-type identities and undergo dynamic transitions during brain development. These networks are highly dynamic during development and over the course of evolution. One such interaction is between radial-glia-enriched ORC4 and miR-2115, a great-ape-specific miRNA, which appears to control radial glia proliferation rates during human brain development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41593-018-0265-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312854PMC
December 2018

Identification of cell types in a mouse brain single-cell atlas using low sampling coverage.

BMC Biol 2018 10 11;16(1):113. Epub 2018 Oct 11.

Department of Neurology, UCSF, San Francisco, USA.

Background: High throughput methods for profiling the transcriptomes of single cells have recently emerged as transformative approaches for large-scale population surveys of cellular diversity in heterogeneous primary tissues. However, the efficient generation of such atlases will depend on sufficient sampling of diverse cell types while remaining cost-effective to enable a comprehensive examination of organs, developmental stages, and individuals.

Results: To examine the relationship between sampled cell numbers and transcriptional heterogeneity in the context of unbiased cell type classification, we explored the population structure of a publicly available 1.3 million cell dataset from E18.5 mouse brain and validated our findings in published data from adult mice. We propose a computational framework for inferring the saturation point of cluster discovery in a single-cell mRNA-seq experiment, centered around cluster preservation in downsampled datasets. In addition, we introduce a "complexity index," which characterizes the heterogeneity of cells in a given dataset. Using Cajal-Retzius cells as an example of a limited complexity dataset, we explored whether the detected biological distinctions relate to technical clustering. Surprisingly, we found that clustering distinctions carrying biologically interpretable meaning are achieved with far fewer cells than the originally sampled, though technical saturation of rare populations such as Cajal-Retzius cells is not achieved. We additionally validated these findings with a recently published atlas of cell types across mouse organs and again find using subsampling that a much smaller number of cells recapitulates the cluster distinctions of the complete dataset.

Conclusions: Together, these findings suggest that most of the biologically interpretable cell types from the 1.3 million cell database can be recapitulated by analyzing 50,000 randomly selected cells, indicating that instead of profiling few individuals at high "cellular coverage," cell atlas studies may instead benefit from profiling more individuals, or many time points at lower cellular coverage and then further enriching for populations of interest. This strategy is ideal for scenarios where cost and time are limited, though extremely rare populations of interest (< 1%) may be identifiable only with much higher cell numbers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12915-018-0580-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180488PMC
October 2018

Transcriptional fates of human-specific segmental duplications in brain.

Genome Res 2018 10 18;28(10):1566-1576. Epub 2018 Sep 18.

Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington 98195, USA.

Despite the importance of duplicate genes for evolutionary adaptation, accurate gene annotation is often incomplete, incorrect, or lacking in regions of segmental duplication. We developed an approach combining long-read sequencing and hybridization capture to yield full-length transcript information and confidently distinguish between nearly identical genes/paralogs. We used biotinylated probes to enrich for full-length cDNA from duplicated regions, which were then amplified, size-fractionated, and sequenced using single-molecule, long-read sequencing technology, permitting us to distinguish between highly identical genes by virtue of multiple paralogous sequence variants. We examined 19 gene families as expressed in developing and adult human brain, selected for their high sequence identity (average >99%) and overlap with human-specific segmental duplications (SDs). We characterized the transcriptional differences between related paralogs to better understand the birth-death process of duplicate genes and particularly how the process leads to gene innovation. In 48% of the cases, we find that the expressed duplicates have changed substantially from their ancestral models due to novel sites of transcription initiation, splicing, and polyadenylation, as well as fusion transcripts that connect duplication-derived exons with neighboring genes. We detect unannotated open reading frames in genes currently annotated as pseudogenes, while relegating other duplicates to nonfunctional status. Our method significantly improves gene annotation, specifically defining full-length transcripts, isoforms, and open reading frames for new genes in highly identical SDs. The approach will be more broadly applicable to genes in structurally complex regions of other genomes where the duplication process creates novel genes important for adaptive traits.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.237610.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169893PMC
October 2018
-->