miR-7 controls glutamatergic transmission and neuronal connectivity in a Cdr1as-dependent manner.

The circular RNA (circRNA) Cdr1as is conserved across mammals and highly expressed in neurons, where it directly interacts with microRNA miR-7. However, the biological function of this interaction is unknown. Here, using primary cortical murine neurons, we demonstrate that stimulating neurons by sustained depolarization rapidly induces two-fold transcriptional upregulation of Cdr1as and strong post-transcriptional stabilization of miR-7. Cdr1as loss causes doubling of glutamate release from stimulated synapses and increased frequency and duration of local neuronal bursts. Moreover, the periodicity of neuronal networks increases, and synchronicity is impaired. Strikingly, these effects are reverted by sustained expression of miR-7, which also clears Cdr1as molecules from neuronal projections. Consistently, without Cdr1as, transcriptomic changes caused by miR-7 overexpression are stronger (including miR-7-targets downregulation) and enriched in secretion/synaptic plasticity pathways. Altogether, our results suggest that in cortical neurons Cdr1as buffers miR-7 activity to control glutamatergic excitatory transmission and neuronal connectivity important for long-lasting synaptic adaptations.

Open-ST: High-resolution spatial transcriptomics in 3D.

Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.

Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids.

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene expression profiles of individual cells, allowing the capture of cellular diversity. In order to overcome limitations posed by difficult-to-isolate cell types, an alternative approach aiming at recovering single nuclei instead of intact cells can be utilized for sequencing, making transcriptome profiling of individual cells universally applicable. These techniques have become a cornerstone in the study of brain organoids, establishing them as models of the developing human brain. Leveraging the potential of single-cell and single-nucleus transcriptomics in brain organoid research, this protocol presents a step-by-step guide encompassing key procedures such as organoid dissociation, single-cell or nuclei isolation, library preparation and sequencing. By implementing these alternative approaches, researchers can obtain high-quality datasets, enabling the identification of neuronal and non-neuronal cell types, gene expression profiles, and cell lineage trajectories. This facilitates comprehensive investigations into cellular processes and molecular mechanisms shaping brain development.

ciRS-7 and miR-7 regulate ischemia-induced neuronal death via glutamatergic signaling.

Brain functionality relies on finely tuned regulation of gene expression by networks of non-coding RNAs (ncRNAs) such as the one composed by the circular RNA ciRS-7 (also known as CDR1as), the microRNA miR-7, and the long ncRNA Cyrano. We describe ischemia-induced alterations in the ncRNA network both in vitro and in vivo and in transgenic mice lacking ciRS-7 or miR-7. Our data show that cortical neurons downregulate ciRS-7 and Cyrano and upregulate miR-7 expression during ischemia. Mice lacking ciRS-7 exhibit reduced lesion size and motor impairment, while the absence of miR-7 alone results in increased ischemia-induced neuronal death. Moreover, miR-7 levels in pyramidal excitatory neurons regulate neurite morphology and glutamatergic signaling, suggesting a potential molecular link to the in vivo phenotype. Our data reveal the role of ciRS-7 and miR-7 in modulating ischemic stroke outcome, shedding light on the pathophysiological function of intracellular ncRNA networks in the brain.

Spatiotemporal, optogenetic control of gene expression in organoids.

Organoids derived from stem cells have become an increasingly important tool for studying human development and modeling disease. However, methods are still needed to control and study spatiotemporal patterns of gene expression in organoids. Here we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes in programmable spatiotemporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. Spatial and single-cell transcriptomic analyses showed that this local induction was sufficient to generate stereotypically patterned organoids and revealed new insights into SHH's contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.

Defining the landscape of circular RNAs in neuroblastoma unveils a global suppressive function of MYCN.

Circular RNAs (circRNAs) are a regulatory RNA class. While cancer-driving functions have been identified for single circRNAs, how they modulate gene expression in cancer is not well understood. We investigate circRNA expression in the pediatric malignancy, neuroblastoma, through deep whole-transcriptome sequencing in 104 primary neuroblastomas covering all risk groups. We demonstrate that MYCN amplification, which defines a subset of high-risk cases, causes globally suppressed circRNA biogenesis directly dependent on the DHX9 RNA helicase. We detect similar mechanisms in shaping circRNA expression in the pediatric cancer medulloblastoma implying a general MYCN effect. Comparisons to other cancers identify 25 circRNAs that are specifically upregulated in neuroblastoma, including circARID1A. Transcribed from the ARID1A tumor suppressor gene, circARID1A promotes cell growth and survival, mediated by direct interaction with the KHSRP RNA-binding protein. Our study highlights the importance of MYCN regulating circRNAs in cancer and identifies molecular mechanisms, which explain their contribution to neuroblastoma pathogenesis.

Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids.

Herpes simplex encephalitis is a life-threatening disease of the central nervous system caused by herpes simplex viruses (HSVs). Following standard of care with antiviral acyclovir treatment, most patients still experience various neurological sequelae. Here we characterize HSV-1 infection of human brain organoids by combining single-cell RNA sequencing, electrophysiology and immunostaining. We observed strong perturbations of tissue integrity, neuronal function and cellular transcriptomes. Under acyclovir treatment viral replication was stopped, but did not prevent HSV-1-driven defects such as damage of neuronal processes and neuroepithelium. Unbiased analysis of pathways deregulated upon infection revealed tumour necrosis factor activation as a potential causal factor. Combination of anti-inflammatory drugs such as necrostatin-1 or bardoxolone methyl with antiviral treatment prevented the damages caused by infection, indicating that tuning the inflammatory response in acute infection may improve current therapeutic strategies.

Establishment of gastrointestinal assembloids to study the interplay between epithelial crypts and their mesenchymal niche.

The cellular organization of gastrointestinal crypts is orchestrated by different cells of the stromal niche but available in vitro models fail to fully recapitulate the interplay between epithelium and stroma. Here, we establish a colon assembloid system comprising the epithelium and diverse stromal cell subtypes. These assembloids recapitulate the development of mature crypts resembling in vivo cellular diversity and organization, including maintenance of a stem/progenitor cell compartment in the base and their maturation into secretory/absorptive cell types. This process is supported by self-organizing stromal cells around the crypts that resemble in vivo organization, with cell types that support stem cell turnover adjacent to the stem cell compartment. Assembloids that lack BMP receptors either in epithelial or stromal cells fail to undergo proper crypt formation. Our data highlight the crucial role of bidirectional signaling between epithelium and stroma, with BMP as a central determinant of compartmentalization along the crypt axis.

Prox2 and Runx3 vagal sensory neurons regulate esophageal motility.

Vagal sensory neurons monitor mechanical and chemical stimuli in the gastrointestinal tract. Major efforts are underway to assign physiological functions to the many distinct subtypes of vagal sensory neurons. Here, we use genetically guided anatomical tracing, optogenetics, and electrophysiology to identify and characterize vagal sensory neuron subtypes expressing Prox2 and Runx3 in mice. We show that three of these neuronal subtypes innervate the esophagus and stomach in regionalized patterns, where they form intraganglionic laminar endings. Electrophysiological analysis revealed that they are low-threshold mechanoreceptors but possess different adaptation properties. Lastly, genetic ablation of Prox2 and Runx3 neurons demonstrated their essential roles for esophageal peristalsis in freely behaving mice. Our work defines the identity and function of the vagal neurons that provide mechanosensory feedback from the esophagus to the brain and could lead to better understanding and treatment of esophageal motility disorders.

Sites of transcription initiation drive mRNA isoform selection.

The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3' end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription initiation (TSS). "Dominant promoters," characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.

Single-cell and spatial transcriptomics: deciphering brain complexity in health and disease.

In the past decade, single-cell technologies have proliferated and improved from their technically challenging beginnings to become common laboratory methods capable of determining the expression of thousands of genes in thousands of cells simultaneously. The field has progressed by taking the CNS as a primary research subject - the cellular complexity and multiplicity of neuronal cell types provide fertile ground for the increasing power of single-cell methods. Current single-cell RNA sequencing methods can quantify gene expression with sufficient accuracy to finely resolve even subtle differences between cell types and states, thus providing a great tool for studying the molecular and cellular repertoire of the CNS and its disorders. However, single-cell RNA sequencing requires the dissociation of tissue samples, which means that the interrelationships between cells are lost. Spatial transcriptomic methods bypass tissue dissociation and retain this spatial information, thereby allowing gene expression to be assessed across thousands of cells within the context of tissue structural organization. Here, we discuss how single-cell and spatially resolved transcriptomics have been contributing to unravelling the pathomechanisms underlying brain disorders. We focus on three areas where we feel these new technologies have provided particularly useful insights: selective neuronal vulnerability, neuroimmune dysfunction and cell-type-specific treatment response. We also discuss the limitations and future directions of single-cell and spatial RNA sequencing technologies.

C. elegans Gene Regulatory Alleles and Reporter Bashing Studies.

Gene regulation has been studied in C. elegans for over 30 years. In this analysis of 102 publications, we find that most transcriptional cis-regulatory elements are located within 5,000 bp of the transcription start site. Over 75% of studies conclude that transcriptional elements and 5'UTRs activate-, while 3'UTRs repress gene expression. While gene regulatory mutations make up less than 0.8% of alleles in forward genetics screens, recent CRISPR-Cas approaches are increasing the number of tested mutations. This work provides a resource of known gene regulatory sequences in C.elegans .

A Circular RNA Expressed from the FAT3 Locus Regulates Neural Development.

Circular RNAs (circRNAs) are key regulators of cellular processes, are abundant in the nervous system, and have putative regulatory roles during neural differentiation. However, the knowledge about circRNA functions in brain development is limited. Here, using RNA-sequencing, we show that circRNA levels increased substantially over the course of differentiation of human embryonic stem cells into rostral and caudal neural progenitor cells (NPCs), including three of the most abundant circRNAs, ciRS-7, circRMST, and circFAT3. Knockdown of circFAT3 during early neural differentiation resulted in minor transcriptional alterations in bulk RNA analysis. However, single-cell transcriptomics of 30 and 90 days differentiated cerebral organoids deficient in circFAT3 showed a loss of telencephalic radial glial cells and mature cortical neurons, respectively. Furthermore, non-telencephalic NPCs in cerebral organoids showed changes in the expression of genes involved in neural differentiation and migration, including FAT4, ERBB4, UNC5C, and DCC. In vivo depletion of circFat3 in mouse prefrontal cortex using in utero electroporation led to alterations in the positioning of the electroporated cells within the neocortex. Overall, these findings suggest a conserved role for circFAT3 in neural development involving the formation of anterior cell types, neuronal differentiation, or migration.

Rapid nuclear deadenylation of mammalian messenger RNA.

Poly(A) tails protect RNAs from degradation and their deadenylation rates determine RNA stability. Although poly(A) tails are generated in the nucleus, deadenylation of tails has mostly been investigated within the cytoplasm. Here, we combined long-read sequencing with metabolic labeling, splicing inhibition and cell fractionation experiments to quantify, separately, the genesis and trimming of nuclear and cytoplasmic tails in vitro and in vivo. We present evidence for genome-wide, nuclear synthesis of tails longer than 200 nt, which are rapidly shortened after transcription. Our data suggests that rapid deadenylation is a nuclear process, and that different classes of transcripts and even transcript isoforms have distinct nuclear tail lengths. For example, many long-noncoding RNAs retain long poly(A) tails. Modeling deadenylation dynamics predicts nuclear deadenylation about 10 times faster than cytoplasmic deadenylation. In summary, our data suggests that nuclear deadenylation might be a key mechanism for regulating mRNA stability, abundance, and subcellular localization.

SLAM-Drop-seq reveals mRNA kinetic rates throughout the cell cycle.

RNA abundance is tightly regulated in eukaryotic cells by modulating the kinetic rates of RNA production, processing, and degradation. To date, little is known about time­dependent kinetic rates during dynamic processes. Here, we present SLAM­Drop­seq, a method that combines RNA metabolic labeling and alkylation of modified nucleotides in methanol­fixed cells with droplet­based sequencing to detect newly synthesized and preexisting mRNAs in single cells. As a first application, we sequenced 7280 HEK293 cells and calculated gene­specific kinetic rates during the cell cycle using the novel package Eskrate. Of the 377 robust­cycling genes that we identified, only a minor fraction is regulated solely by either dynamic transcription or degradation (6 and 4%, respectively). By contrast, the vast majority (89%) exhibit dynamically regulated transcription and degradation rates during the cell cycle. Our study thus shows that temporally regulated mRNA degradation is fundamental for the correct expression of a majority of cycling genes. SLAM­Drop­seq, combined with Eskrate, is a powerful approach to understanding the underlying mRNA kinetics of single­cell gene expression dynamics in continuous biological processes.

Mechanisms of IRF2BPL-related disorders and identification of a potential therapeutic strategy.

The recently discovered neurological disorder NEDAMSS is caused by heterozygous truncations in the transcriptional regulator IRF2BPL. Here, we reprogram patient skin fibroblasts to astrocytes and neurons to study mechanisms of this newly described disease. While full-length IRF2BPL primarily localizes to the nucleus, truncated patient variants sequester the wild-type protein to the cytoplasm and cause aggregation. Moreover, patient astrocytes fail to support neuronal survival in coculture and exhibit aberrant mitochondria and respiratory dysfunction. Treatment with the small molecule copper ATSM (CuATSM) rescues neuronal survival and restores mitochondrial function. Importantly, the in vitro findings are recapitulated in vivo, where co-expression of full-length and truncated IRF2BPL in Drosophila results in cytoplasmic accumulation of full-length IRF2BPL. Moreover, flies harboring heterozygous truncations of the IRF2BPL ortholog (Pits) display progressive motor defects that are ameliorated by CuATSM treatment. Our findings provide insights into mechanisms involved in NEDAMSS and reveal a promising treatment for this severe disorder.

Long-term imaging reveals behavioral plasticity during C. elegans dauer exit.

BACKGROUND: During their lifetime, animals must adapt their behavior to survive in changing environments. This ability requires the nervous system to undergo adjustments at distinct temporal scales, from short-term dynamic changes in expression of neurotransmitters and receptors to longer-term growth, spatial and connectivity reorganization, while integrating external stimuli. The nematode Caenorhabditis elegans provides a model of nervous system plasticity, in particular its dauer exit decision. Under unfavorable conditions, larvae will enter the non-feeding and non-reproductive stress-resistant dauer stage and adapt their behavior to cope with the harsh new environment, with active reversal under improved conditions leading to resumption of reproductive development. However, how different environmental stimuli regulate the exit decision mechanism and thereby drive the larva's behavioral change is unknown. To fill this gap and provide insights on behavioral changes over extended periods of time, we developed a new open hardware method for long-term imaging (12h) of C. elegans larvae. RESULTS: Our WormObserver platform comprises open hardware and software components for video acquisition, automated processing of large image data (> 80k images/experiment) and data analysis. We identified dauer-specific behavioral motifs and characterized the behavioral trajectory of dauer exit in different environments and genetic backgrounds to identify key decision points and stimuli promoting dauer exit. Combining long-term behavioral imaging with transcriptomics data, we find that bacterial ingestion triggers a change in neuropeptide gene expression to establish post-dauer behavior. CONCLUSIONS: Taken together, we show how a developing nervous system can robustly integrate environmental changes activate a developmental switch and adapt the organism's behavior to a new environment. WormObserver is generally applicable to other research questions within and beyond the C. elegans field, having a modular and customizable character and allowing assessment of behavioral plasticity over longer periods.

MicroRNAs are deeply linked to the emergence of the complex octopus brain.

Soft-bodied cephalopods such as octopuses are exceptionally intelligent invertebrates with a highly complex nervous system that evolved independently from vertebrates. Because of elevated RNA editing in their nervous tissues, we hypothesized that RNA regulation may play a major role in the cognitive success of this group. We thus profiled messenger RNAs and small RNAs in three cephalopod species including 18 tissues of the Octopus vulgaris. We show that the major RNA innovation of soft-bodied cephalopods is an expansion of the microRNA (miRNA) gene repertoire. These evolutionarily novel miRNAs were primarily expressed in adult neuronal tissues and during the development and had conserved and thus likely functional target sites. The only comparable miRNA expansions happened, notably, in vertebrates. Thus, we propose that miRNAs are intimately linked to the evolution of complex animal brains.

Cell type diversity in a developing octopus brain.

Octopuses are mollusks that have evolved intricate neural systems comparable with vertebrates in terms of cell number, complexity and size. The brain cell types that control their sophisticated behavioral repertoire are still unknown. Here, we profile the cell diversity of the paralarval Octopus vulgaris brain to build a cell type atlas that comprises mostly neural cells, but also multiple glial subtypes, endothelial cells and fibroblasts. We spatially map cell types to the vertical, subesophageal and optic lobes. Investigation of cell type conservation reveals a shared gene signature between glial cells of mouse, fly and octopus. Genes related to learning and memory are enriched in vertical lobe cells, which show molecular similarities with Kenyon cells in Drosophila. We construct a cell type taxonomy revealing transcriptionally related cell types, which tend to appear in the same brain region. Together, our data sheds light on cell type diversity and evolution in the octopus brain.