Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex

Mich, John K.; Graybuck, Lucas T.; Hess, Erik E.; Mahoney, Joseph T.; Kojima, Yoshiyuki; Ding, Yi; Somasundaram, Saroja; Miller, Jeremy A.; Weed, Natalie; Omstead, Victoria; Bishaw, Yemeserach; Shapovalova, Nadiya V.; Martinez, Refugio A.; Fong, Olivia; Yao, Shenqin; Mortrud, Marty; Chong, Peter Han Joo; Loftus, Luke V.; Bertagnolli, Darren; Goldy, Jeff; Casper, Tamara; Dee, Nick; Opitz-Araya, Ximena; Çetin, Ali; Smith, Kimberly A.; Gwinn, Ryder P.; Cobbs, Charles; Ojemann, Jeffrey G.; Keene, C. Dirk; Silbergeld, Daniel L.; Sunkin, Susan M.; Gradinaru, Viviana; Horwitz, Gregory D.; Zeng, Hongkui; Tasic, Bosiljka; Lein, Ed; Ting, Jonathan T.

doi:10.1101/555318

Cited by 21 publications

(25 citation statements)

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“…In order to determine the fidelity of organoids as a model for the epigenomic landscape of the developing brain, functional characterization of conserved and non-conserved chromatin features in vivo and in vitro will be required. In addition to serving as a reference for evaluating in vitro models of human brain development, our data will enable scalable prediction of candidate cell type specific regulatory elements that could facilitate the development of enhancer-based tools for accessing molecularly-defined cell types [57][58][59] .…”

Section: Discussionmentioning

confidence: 99%

Single cell epigenomic atlas of the developing human brain and organoids

Rs¹,

Wilfert

et al. 2019

Preprint

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31Dynamic changes in chromatin accessibility coincide with important aspects of neuronal differentiation, such as 32 fate specification and arealization and confer cell type-specific associations to neurodevelopmental disorders. 33However, studies of the epigenomic landscape of the developing human brain have yet to be performed at single-34 cell resolution. Here, we profiled chromatin accessibility of >75,000 cells from eight distinct areas of developing 35human forebrain using single cell ATAC-seq (scATACseq). We identified thousands of loci that undergo 36 extensive cell type-specific changes in accessibility during corticogenesis. Chromatin state profiling also reveals 37 novel distinctions between neural progenitor cells from different cortical areas not seen in transcriptomic profiles 38 and suggests a role for retinoic acid signaling in cortical arealization. Comparison of the cell type-specific 39 chromatin landscape of cerebral organoids to primary developing cortex found that organoids establish broad 40 cell type-specific enhancer accessibility patterns similar to the developing cortex, but lack many putative 41 regulatory elements identified in homologous primary cell types. Together, our results reveal the important 42 contribution of chromatin state to the emerging patterns of cell type diversity and cell fate specification and 43 provide a blueprint for evaluating the fidelity and robustness of cerebral organoids as a model for cortical 44 development. 45 46Main text 47The diverse cell types of the human cerebral cortex (Fig. 1a) have been mostly classified based on a handful of 48 morphological, anatomical, and physiological features. Recent innovations in single cell genomics, such as single 49 cell mRNA sequencing (scRNA-seq), have enabled massively parallel profiling of thousands of molecular 50 features in every cell, uncovering the remarkable molecular diversity of cell types previously considered 51 homologous, such as excitatory neurons located in different areas of the cerebral cortex 1-6 . However, the 52 developmental mechanisms underlying the emergence of distinct cellular identities are largely unknown, as most 53 cortical neurons are generated at stages that are inaccessible to experimentation 5 . 54 55Over 60 years ago, Conrad Waddington introduced the concept of an epigenomic landscape to account for the 56 emergence of distinct cell fates 7 . In particular, chromatin state defines the functional architecture of the genome 57

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Section: Discussionmentioning

confidence: 99%

Single cell epigenomic atlas of the developing human brain and organoids

Rs¹,

Wilfert

et al. 2019

Preprint

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“…To overcome the difficulty in targeting diverse interneuron subclasses, we performed rapid viral genetic labeling of cortical GABAergic interneurons. We applied an adeno-associated virus (AAV) vector that drives SYFP2 reporter express under the control of an optimized forebrain GABAergic neuron enhancer (Stuhmer et al, 2002; Dimidschstein et al, 2016) in human organotypic slice culture (see Methods ; Ting et al, 2018; Mich et al, 2020). Inhibitory subclass identity was established post-hoc by mFISH staining.…”

Section: Resultsmentioning

confidence: 99%

“…We showed that this methodological approach of quadruple modality functional characterization can be extended to slice culture with viral labeling as well. This approach is well poised to take advantage of an increasing number of enhancer-driven subclass and type-specific viral tools (Dimidschstein et al, 2016; Hrvatin et al, 2019; Jüttner et al, 2019; Mehta et al, 2019; Nair et al, 2019; Mich et al, 2020; Graybuck et al, 2020; Vormstein-Schneider et al, 2020) to allow prospective targeting of cell classes or subclasses, and subsequent refinement of cell type identity by a combination of marker genes using mFISH. Brain slice culturing and viral transgenesis will allow not only measurement, but also functional manipulation of the human microcircuit, e.g.…”

Section: Discussionmentioning

confidence: 99%

“…These excised tissues are often distal to epileptic seizure foci or deep brain tumors and in many cases are devoid of overt pathology (Szabadics et al, 2006; Verhoog et al, 2013; Tian et al, 2014; Wang et al, 2015; Beaulieu-Laroche et al, 2018; Berg et al 2020). Human ex vivo neocortical slices can also be cultured for weeks to months (Eugene et al, 2014; Schwarz et al, 2017), and viral transgenesis allows genetic manipulation of cells in brain slices (Andersson et al, 2016; Le Duigou et al, 2018; Ting et al, 2018; Mich et al, 2020; Schwarz et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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Target cell-specific synaptic dynamics of excitatory to inhibitory neuron connections in supragranular layers of human neocortex

Kim

Radaelli

Thomsen

et al. 2020

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Prospective and post-hoc molecular identification of specific neuron types is essential for functional studies of cellular and synaptic properties. We demonstrate a thick brain slice mFISH technique applied to multi-patch-clamp recordings in human cortical slices obtained from neurosurgical-excised tissue to reveal the molecular and morpho-electric properties of synaptically connected neurons, both with and without prospective AAV based genetic labeling. This quadruple modality methodology should be extensible to other local brain circuits in many organisms.

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“…In one of these studies, single-cell ATAC-Seq paired with single-cell RNAseq was employed in order to detect which GREs are active in each cluster identified. They created AAVs with the selected GREs driving expression of recombinases or fluorescent proteins and validated their expression [358], [359].…”

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confidence: 99%

Molecular profiling and targeting of brain cells and circuits related to energy balance and food reward

Georgiadou¹

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In the great, grand scheme of things, we're just tiny specks that will one day be forgotten. So it doesn't matter what we did in the past or how we'll be remembered. The only thing that matters is right now, this moment. This one spectacular moment we are sharing together. Molecular profiling and genetic targeting of brain cells RNA sequencing for molecular profiling and classification of brain cells A popular and reliable method to investigate brain heterogeneity is RNA sequencing, which provides information about the whole transcriptome and can be applied to a brain region, a specific cell population or at the single cell level. Many different cell dissociation protocols and sequencing technologies exist, the application of which can vary depending on the brain region, cell abundance, developmental stage and the desired sequencing depth. For example, when researchers desire to sequence a particular cell type, they can use a transgenic animal expressing a fluorescent protein in the cell population of interest, Targeting neural subtypes and circuits with viral vectors Molecular profiling of cells is very useful for identifying novel cell types and gaining insights into their molecular makeup and transcriptional responses to cues. Nevertheless, in order to understand cell types better, we need to gain genetic access to them. A popular method for gene delivery into the brain in order to map cells, visualize brain connectivity and study the function of cell populations and circuits is viral vector technology. Viral vectors are used to target genetic information into cells of choice. To this end, either wild-type or engineered viral capsids are used to "package" the genetic information (transgene) designed in a way to facilitate proper expression of RNA or protein. Few of the most commonly used viral vectors in neuroscience are the adeno-associated virus (AAV), lentivirus (LV), canine adenovirus 2 (Cav2) and rabies. The two former are mostly used for anterograde targeting, while the two latter are used for retrograde targeting of neurons and are extremely useful for brain mapping of circuits. In this thesis we use AAV and Cav2, therefore we will discuss those. Adeno-associated viral vectors (AAVs) AAV is the most popular viral vector system used in neuroscience and gene therapy. The first gene therapy drug, which treats retinal dystrophy, was approved in 2017, while there are at least 20 approved gene therapy treatments available and at least 4000 ongoing clinical trials [25], [26]. Besides the fact that the AAV capsid has limited capacity and can accommodate only up to 5.2 kb of DNA, their use has a lot of advantages. Genes delivered by AAVs do not incorporate into the host genome and rather stay episomal [27], therefore they do not cause insertional mutations that could lead to toxic consequences. AAV capsids Hypothalamus, energy balance and leptin In this thesis we mostly focus on cells located in the hypothalamus (Chapters 1-3) and profile leptin receptor-expressing cells (Chapters 1 & 2). The hypothalamus is loc...

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Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex

Cited by 21 publications

References 60 publications

Single cell epigenomic atlas of the developing human brain and organoids

Single cell epigenomic atlas of the developing human brain and organoids

Target cell-specific synaptic dynamics of excitatory to inhibitory neuron connections in supragranular layers of human neocortex

Molecular profiling and targeting of brain cells and circuits related to energy balance and food reward

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