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MOTIVATION: Imaging Spatial Transcriptomics techniques characterize gene expression in cells in their native context by imaging barcoded probes for mRNA with single molecule resolution. However, the need to acquire many rounds of high-magnification imaging data limits the throughput and impact of existing methods. RESULTS: We describe the Joint Sparse method for Imaging Transcriptomics, an algorithm for decoding lower magnification Imaging Spatial Transcriptomics data than that used in standard experimental workflows. Joint Sparse method for Imaging Transcriptomics incorporates codebook knowledge and sparsity assumptions into an optimization problem, which is less reliant on well separated optical signals than current pipelines. Using experimental data obtained by performing Multiplexed Error-Robust Fluorescence in situ Hybridization on tissue from mouse brain, we demonstrate that Joint Sparse method for Imaging Transcriptomics enables improved throughput and recovery performance over standard decoding methods. AVAILABILITY AND IMPLEMENTATION: Software implementation of JSIT, together with example files, is available at https://github.com/jpbryan13/JSIT.
Asunto(s)
Perfilación de la Expresión Génica , Transcriptoma , Animales , Ratones , Hibridación Fluorescente in Situ/métodos , Perfilación de la Expresión Génica/métodos , Programas Informáticos , AlgoritmosRESUMEN
Serine/Threonine Kinase 11 (STK11) encodes an important tumor suppressor that is frequently mutated in lung adenocarcinoma. Clinical studies have shown that mutations in STK11 resulting in loss of function correlate with resistance to anti-PD-1 monoclonal antibody therapy in KRAS-driven non-small cell lung cancer (NSCLC), but the molecular mechanisms responsible remain unclear. Despite this uncertainty, STK11 functional status is emerging as a reliable biomarker for predicting non-response to anti-PD-1 therapy in NSCLC patients. The clinical utility of this biomarker ultimately depends upon accurate classification of STK11 variants. For nonsense variants occurring early in the STK11 coding region, this assessment is straightforward. However, rigorously demonstrating the functional impact of missense variants remains an unmet challenge. Here we present data characterizing four STK11 splice-site variants by analyzing tumor mRNA, and 28 STK11 missense variants using an in vitro kinase assay combined with a cell-based p53-dependent luciferase reporter assay. The variants we report were identified in primary human NSCLC biopsies in collaboration with the University of Vermont Genomic Medicine group. Additionally, we compare our experimental results with data from 22 in silico predictive algorithms. Our work highlights the power, utility and necessity of functional variant assessment and will aid STK11 variant curation, provide a platform to assess novel STK11 variants and help guide anti-PD-1 therapy utilization in KRAS-driven NSCLCs.
Asunto(s)
Quinasas de la Proteína-Quinasa Activada por el AMP/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Mutación , Quinasas de la Proteína-Quinasa Activada por el AMP/metabolismo , Empalme Alternativo , Biomarcadores de Tumor , Sistemas CRISPR-Cas , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Análisis Mutacional de ADN , Susceptibilidad a Enfermedades , Edición Génica , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/mortalidad , Mutagénesis Sitio-Dirigida , Mutación Missense , Fosforilación , Pronóstico , Sitios de Empalme de ARNRESUMEN
A long CGG-repeat tract in the FMR1 gene induces the epigenetic silencing that causes fragile X syndrome (FXS). Epigenetic changes include H4K20 trimethylation, a heterochromatic modification frequently implicated in transcriptional silencing. Here, we report that treatment with A-196, an inhibitor of SUV420H1/H2, the enzymes responsible for H4K20 di-/trimethylation, does not affect FMR1 transcription, but does result in increased chromosomal duplications. Increased duplications were also seen in FXS cells treated with SCR7, an inhibitor of Lig4, a ligase essential for NHEJ. Our study suggests that the fragile X (FX) locus is prone to spontaneous DNA damage that is normally repaired by NHEJ. We suggest that heterochromatinization of the FX allele may be triggered, at least in part, in response to this DNA damage.
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The mammalian cerebral cortex comprises a complex neuronal network that maintains a delicate balance between excitatory neurons and inhibitory interneurons. Previous studies, including our own research, have shown that specific interneuron subtypes are closely associated with particular pyramidal neuron types, forming stereotyped local inhibitory microcircuits. However, the developmental processes that establish these precise networks are not well understood. Here we show that pyramidal neuron types are instrumental in driving the terminal differentiation and maintaining the survival of specific associated interneuron subtypes. In a wild-type cortex, the relative abundance of different interneuron subtypes aligns precisely with the pyramidal neuron types to which they synaptically target. In Fezf2 mutant cortex, characterized by the absence of layer 5 pyramidal tract neurons and an expansion of layer 6 intratelencephalic neurons, we observed a corresponding decrease in associated layer 5b interneurons and an increase in layer 6 subtypes. Interestingly, these shifts in composition are achieved through mechanisms specific to different interneuron types. While SST interneurons adjust their abundance to the change in pyramidal neuron prevalence through the regulation of programmed cell death, parvalbumin interneurons alter their identity. These findings illustrate two key strategies by which the dynamic interplay between pyramidal neurons and interneurons allows local microcircuits to be sculpted precisely. These insights underscore the precise roles of extrinsic signals from pyramidal cells in the establishment of interneuron diversity and their subsequent integration into local cortical microcircuits.
RESUMEN
The mammalian cerebral cortex comprises a complex neuronal network that maintains a delicate balance between excitatory neurons and inhibitory interneurons. Previous studies, including our own research, have shown that specific interneuron subtypes are closely associated with particular pyramidal neuron types, forming stereotyped local inhibitory microcircuits. However, the developmental processes that establish these precise networks are not well understood. Here we show that pyramidal neuron types are instrumental in driving the terminal differentiation and maintaining the survival of specific associated interneuron subtypes. In a wild-type cortex, the relative abundance of different interneuron subtypes aligns precisely with the pyramidal neuron types to which they synaptically target. In Fezf2 mutant cortex, characterized by the absence of layer 5 pyramidal tract neurons and an expansion of layer 6 intratelencephalic neurons, we observed a corresponding decrease in associated layer 5b interneurons and an increase in layer 6 subtypes. Interestingly, these shifts in composition are achieved through mechanisms specific to different interneuron types. While SST interneurons adjust their abundance to the change in pyramidal neuron prevalence through the regulation of programmed cell death, parvalbumin interneurons alter their identity. These findings illustrate two key strategies by which the dynamic interplay between pyramidal neurons and interneurons allows local microcircuits to be sculpted precisely. These insights underscore the precise roles of extrinsic signals from pyramidal cells in the establishment of interneuron diversity and their subsequent integration into local cortical microcircuits.
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Ovulation is a spatiotemporally coordinated process that involves several tightly controlled events, including oocyte meiotic maturation, cumulus expansion, follicle wall rupture and repair, and ovarian stroma remodeling. To date, no studies have detailed the precise window of ovulation at single-cell resolution. Here, we performed parallel single-cell RNA-seq and spatial transcriptomics on paired mouse ovaries across an ovulation time course to map the spatiotemporal profile of ovarian cell types. We show that major ovarian cell types exhibit time-dependent transcriptional states enriched for distinct functions and have specific localization profiles within the ovary. We also identified gene markers for ovulation-dependent cell states and validated these using orthogonal methods. Finally, we performed cell-cell interaction analyses to identify ligand-receptor pairs that may drive ovulation, revealing previously unappreciated interactions. Taken together, our data provides a rich and comprehensive resource of murine ovulation that can be mined for discovery by the scientific community.
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More than 100 years since the first description of Chagas Disease and with over 29,000 new cases annually due to vector transmission (in 2010), American Trypanosomiasis remains a Neglected Tropical Disease (NTD). This study presents the most comprehensive Trypanosoma cruzi sampling in terms of geographic locations and triatomine species analyzed to date and includes both nuclear and mitochondrial genomes. This addresses the gap of information from North and Central America. We incorporate new and previously published DNA sequence data from two mitochondrial genes, Cytochrome oxidase II (COII) and NADH dehydrogenase subunit 1 (ND1). These T. cruzi samples were collected over a broad geographic range including 111 parasite DNA samples extracted from triatomines newly collected across North and Central America, all of which were infected with T. cruzi in their natural environment. In addition, we present parasite reduced representation (Restriction site Associated DNA markers, RAD-tag) genomic nuclear data combined with the mitochondrial gene sequences for a subset of the triatomines (27 specimens) collected from Guatemala and El Salvador. Our mitochondrial phylogenetic reconstruction revealed two of the major mitochondrial lineages circulating across North and Central America, as well as the first ever mitochondrial data for TcBat from a triatomine collected in Central America. Our data also show that within mtTcIII, North and Central America represent an independent, distinct clade from South America, named here as mtTcIIINA-CA, geographically restricted to North and Central America. Lastly, the most frequent lineage detected across North and Central America, mtTcI, was also an independent, distinct clade from South America, noted as mtTcINA-CA. Furthermore, nuclear genome data based on Single Nucleotide Polymorphism (SNP) showed genetic structure of lineage TcI from specimens collected in Guatemala and El Salvador supporting the hypothesis that genetic diversity at a local scale has a geographical component. Our multiscale analysis contributes to the understanding of the independent and distinct evolution of T. cruzi lineages in North and Central America regions.