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Mesoscale imaging, inactivation, and collaboration in a standardized visual decision-making task
Full text linkTypical neuroscience experiments in the field of decision-making have focused on recording in one or a few brain areas while animals perform a task created by and only used in their own lab with their own custom hardware and software. This makes combining information from these studies to address big questions, such as how information across the whole brain produces a decision, impossible. Big questions such as this are better addressed by collaborations that can enforce standardized methods across their members and can therefore pool their efforts and resources. One such collaboration, the IBL, is focusing on how the mouse brain solves a basic perceptual decision-making task. Towards this goal, in collaboration with the IBL, I have created a standardized visual decision-making task that allows
comparison of experiments both between labs and between experimental modalities. I have recorded thousands of neurons across the brain in contribution to a brainwide map of single cell spiking activity during decision-making. These recordings are in contribution to three main scientific goals, a brainwide map of activity related to decision-making, methods for standardized and reproducible electrophysiology recordings, and an electrophysiological atlas of the mouse brain. In my own experiments performed
alongside those of the IBL, I used an unbiased inhibition scan across the dorsal cortex to determine the causal cortical areas for performing the IBL task. This revealed that visual cortex inactivation impairs accumulation of contralateral visual information, and secondary motor cortex inactivation biases the starting point of the decision process away from the contralateral side. I additionally performed calcium imaging of the whole mouse dorsal cortex as an independent source of neural recording from the IBL
brainwide map. These recordings indicate that task information is broadcast widely across the cortical network, but the strongest information is localized in the expected nodes: initially after stimulus onset in V1, then progressively in M2. These recordings additionally revealed two cortical representations of stimulus or choice expectation: a selective prestimulus suppression, and post stimulus excitation for the expected stimulus-driven areas in V1 and M2, and a potential embodied expectation that is reflected in the paw and torso somatosensory areas. Finally I have shown proof-of-principle experiments that one can
simultaneously optogenetically manipulate targeted cortical areas, while monitoring the neural activity across the whole dorsal cortex with widefield calcium imaging. Though this experiment still has a few flaws such as the visual detection of red light, it could lead to many discoveries about the basic function of cortical networks
Genome-wide association analysis and replication in 810,625 individuals with varicose veins
Full text linkVaricose veins affect one-third of Western society, with a significant subset of patients developing venous ulceration, costing $14.9 billion annually in the USA. Current management consists of either compression stockings, or surgical ablation for more advanced disease. Most varicose veins patients report a positive family history, and heritability is ~17%. We describe the largest two-stage genome-wide association study of varicose veins in 401,656 individuals from UK Biobank, and replication in 408,969 individuals from 23andMe (total 135,514 cases and 675,111 controls). Forty-nine signals at 46 susceptibility loci were discovered. We map 237 genes to these loci, several of which are biologically plausible and tractable to therapeutic targeting. Pathway analysis identified enrichment in extracellular matrix biology, inflammation, (lymph)angiogenesis, vascular smooth muscle cell migration, and apoptosis. Using a polygenic risk score (PRS) derived in an independent cohort, we demonstrate its predictive utility and correlation with varicose veins surgery
Quantitative relationship between cerebrovascular network and neuronal cell types in mice
Full text linkThe cerebrovasculature and its mural cells must meet brain regional energy demands, but how their spatial relationship with different neuronal cell types varies across the brain remains largely unknown. Here we apply brain-wide mapping methods to comprehensively define the quantitative relationships between the cerebrovasculature, capillary pericytes, and glutamatergic and GABAergic neurons, including neuronal nitric oxide synthase-positive (nNOS+) neurons and their subtypes in adult mice. Our results show high densities of vasculature with high fluid conductance and capillary pericytes in primary motor sensory cortices compared with association cortices that show significant positive and negative correlations with energy-demanding parvalbumin+ and vasomotor nNOS+ neurons, respectively. Thalamo-striatal areas that are connected to primary motor sensory cortices also show high densities of vasculature and pericytes, suggesting dense energy support for motor sensory processing areas. Our cellular-resolution resource offers opportunities to examine spatial relationships between the cerebrovascular network and neuronal cell composition in largely understudied subcortical areas
Epigenetic Mechanisms of Brain Sexual Differentiation
No full textAcross vertebrate species, gonadal hormones coordinate physiology with behavior to facilitate social interactions essential for reproduction and survival. In adulthood, these hormones activate neural circuits that regulate behaviors presenting differently in females and males, such as parenting and territorial aggression. Yet long before sex-typical behaviors emerge at puberty, transient hormone production during sensitive periods of neurodevelopment establish the circuits upon which adult hormones act. How transitory waves of early-life hormone signaling exert lasting effects on the brain remains a central question. Here we discuss how perinatal estradiol signaling organizes cellular and molecular sex differences in the rodent brain. We review classic anatomic studies revealing sex differences in cell number, volume, and neuronal projections, and consider how single-cell sequencing methods enable distinction between sex-biased cell-type abundance and gene expression. Finally, we highlight the recent discovery of a gene regulatory program activated by estrogen receptor α (ERα) following the perinatal hormone surge. A subset of this program displays sustained sex-biased gene expression and chromatin accessibility throughout the postnatal sensitive period, demonstrating a bona fide epigenetic mechanism. We propose that ERα-expressing neurons throughout the social behavior network use similar gene regulatory programs to coordinate brain sexual differentiation
Correction: γδ T cell IFNγ production is directly subverted by Yersinia pseudotuberculosis outer protein YopJ in mice and humans
No full text[This corrects the article DOI: 10.1371/journal.ppat.1010103.]
Longitudinal Intravital Imaging Through Clear Silicone Windows
No full textIntravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse's skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time-up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions
IL-17RA-signaling in Lgr5+ intestinal stem cells induces expression of transcription factor ATOH1 to promote secretory cell lineage commitment
No full textThe Th17 cell-lineage-defining cytokine IL-17A contributes to host defense and inflammatory disease by coordinating multicellular immune responses. The IL-17 receptor (IL-17RA) is expressed by diverse intestinal cell types, and therapies targeting IL-17A induce adverse intestinal events, suggesting additional tissue-specific functions. Here, we used multiple conditional deletion models to identify a role for IL-17A in secretory epithelial cell differentiation in the gut. Paneth, tuft, goblet, and enteroendocrine cell numbers were dependent on IL-17A-mediated induction of the transcription factor ATOH1 in Lgr5+ intestinal epithelial stem cells. Although dispensable at steady state, IL-17RA signaling in ATOH1+ cells was required to regenerate secretory cells following injury. Finally, IL-17A stimulation of human-derived intestinal organoids that were locked into a cystic immature state induced ATOH1 expression and rescued secretory cell differentiation. Our data suggest that the cross talk between immune cells and stem cells regulates secretory cell lineage commitment and the integrity of the mucosa
Pan-human consensus genome significantly improves the accuracy of RNA-seq analyses
Full text linkThe Human Reference Genome serves as the foundation for modern genomic analyses. However, in its present form, it does not adequately represent the vast genetic diversity of the human population. In this study, we explored the consensus genome as a potential successor of the current reference genome and assessed its effect on the accuracy of RNA-seq read alignment. In order to find the best haploid genome representation, we constructed consensus genomes at the pan-human, super-population, and population levels, utilizing variant information from the 1000 Genomes Project. Using personal haploid genomes as the ground truth, we compared mapping errors for real RNA-seq reads aligned to the consensus genomes versus the reference genome. For reads overlapping homozygous variants, we found that the mapping error decreased by a factor of ~2-3 when the reference was replaced with the pan-human consensus genome. We also found that using more population-specific consensuses resulted in little to no increase overusing the pan-human consensus, suggesting a limit in the utility of incorporating more specific genomic variation. Replacing reference with consensus genomes impacts functional analyses, such as differential expressions of isoforms, genes, and splice junctions
Same-Cell Co-Occurrence of RAS Hotspot and BRAF V600E Mutations in Treatment-Naive Colorectal Cancer
No full textPURPOSE: Mitogen-activated protein kinase pathway-activating mutations occur in the majority of colorectal cancer (CRC) cases and show mutual exclusivity. We identified 47 epidermal growth factor receptor/BRAF inhibitor-naive CRC patients with dual RAS hotspot/BRAF V600E mutations (CRC-DD) from a cohort of 4,561 CRC patients with clinical next-generation sequencing results. We aimed to define the molecular phenotypes of the CRC-DD and to test if the dual RAS hotspot/BRAF V600E mutations coexist within the same cell. MATERIALS AND METHODS: We developed a single-cell genotyping method with a mutation detection rate of 96.3% and a genotype prediction accuracy of 92.1%. Mutations in the CRC-DD cohort were analyzed for clonality, allelic imbalance, copy number, and overall survival. RESULTS: Application of single-cell genotyping to four CRC-DD revealed the co-occurrence of both mutations in the following percentages of cells per case: NRAS G13D/KRAS G12C, 95%; KRAS G12D/NRAS G12V, 48%; BRAF V600E/KRAS G12D, 44%; and KRAS G12D/NRAS G13V, 14%, respectively. Allelic imbalance favoring the oncogenic allele was less frequent in CRC-DD (24 of 76, 31.5%, somatic mutations) compared with a curated cohort of CRC with a single-driver mutation (CRC-SD; 119 of 232 mutations, 51.3%; P = .013). Microsatellite instability-high status was enriched in CRC-DD compared with CRC-SD (23% v 11.4%, P = .028). Of the seven CRC-DD cases with multiregional sequencing, five retained both driver mutations throughout all sequenced tumor sites. Both CRC-DD cases with discordant multiregional sequencing were microsatellite instability-high. CONCLUSION: Our findings indicate that dual-driver mutations occur in a rare subset of CRC, often within the same tumor cells and across multiple tumor sites. Their presence and a lower rate of allelic imbalance may be related to dose-dependent signaling within the mitogen-activated protein kinase pathway
A complete reference genome improves analysis of human genetic variation
No full textCompared to its predecessors, the Telomere-to-Telomere CHM13 genome adds nearly 200 million base pairs of sequence, corrects thousands of structural errors, and unlocks the most complex regions of the human genome for clinical and functional study. We show how this reference universally improves read mapping and variant calling for 3202 and 17 globally diverse samples sequenced with short and long reads, respectively. We identify hundreds of thousands of variants per sample in previously unresolved regions, showcasing the promise of the T2T-CHM13 reference for evolutionary and biomedical discovery. Simultaneously, this reference eliminates tens of thousands of spurious variants per sample, including reduction of false positives in 269 medically relevant genes by up to a factor of 12. Because of these improvements in variant discovery coupled with population and functional genomic resources, T2T-CHM13 is positioned to replace GRCh38 as the prevailing reference for human genetics