Imaging cell lineage with a synthetic digital recording system

Cell lineage plays a pivotal role in cell fate determination. Chow et al. demonstrate the use of an integrase-based synthetic barcode system called intMEMOIR, which uses the serine integrase Bxb1 to perform irreversible nucleotide edits. Inducible editing either deletes or inverts its target region, thus encoding information in three-state memory elements, or trits, and avoiding undesired recombination events. Using intMEMOIR combined with single-molecule fluorescence in situ hybridization, the authors were able to identify clonal structures as well as gene expression patterns in the fly brain, enabling both clonal analysis and expression profiling with intact spatial information. The ability to visualize cell lineage relationships directly within their native tissue context provides insights into development and disease

Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH

Visualization of the transcriptome and the nuclear organization in situ has been challenging for single-cell analysis. Here, we demonstrate a multiplexed single-molecule in situ method, intron seqFISH, that allows imaging of 10,421 genes at their nascent transcription active sites in single cells, followed by mRNA and lncRNA seqFISH and immunofluorescence. This nascent transcriptome-profiling method can identify different cell types and states with mouse embryonic stem cells and fibroblasts. The nascent sites of RNA synthesis tend to be localized on the surfaces of chromosome territories, and their organization in individual cells is highly variable. Surprisingly, the global nascent transcription oscillated asynchronously in individual cells with a period of 2 hr in mouse embryonic stem cells, as well as in fibroblasts. Together, spatial genomics of the nascent transcriptome by intron seqFISH reveals nuclear organizational principles and fast dynamics in single cells that are otherwise obscured

Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH

Visualization of the transcriptome and the nuclear organization in situ has been challenging for single-cell analysis. Here, we demonstrate a multiplexed single-molecule in situ method, intron seqFISH, that allows imaging of 10,421 genes at their nascent transcription active sites in single cells, followed by mRNA and lncRNA seqFISH and immunofluorescence. This nascent transcriptome-profiling method can identify different cell types and states with mouse embryonic stem cells and fibroblasts. The nascent sites of RNA synthesis tend to be localized on the surfaces of chromosome territories, and their organization in individual cells is highly variable. Surprisingly, the global nascent transcription oscillated asynchronously in individual cells with a period of 2 hr in mouse embryonic stem cells, as well as in fibroblasts. Together, spatial genomics of the nascent transcriptome by intron seqFISH reveals nuclear organizational principles and fast dynamics in single cells that are otherwise obscured

Imaging cell lineage with a synthetic digital recording system

Multicellular development depends on the differentiation of cells into specific fates with precise spatial organization. Lineage history plays a pivotal role in cell fate decisions, but is inaccessible in most contexts. Engineering cells to actively record lineage information in a format readable in situ would provide a spatially resolved view of lineage in diverse developmental processes. Here, we introduce a serine integrase-based recording system that allows in situ readout, and demonstrate its ability to reconstruct lineage relationships in cultured stem cells and flies. The system, termed intMEMOIR, employs an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. intMEMOIR accurately reconstructed lineage trees in stem cells and enabled simultaneous analysis of single cell clonal history, spatial position, and gene expression in Drosophila brain sections. These results establish a foundation for microscopy-readable clonal analysis and recording in diverse systems

Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+

Imaging the transcriptome in situ with high accuracy has been a major challenge in single-cell biology, which is particularly hindered by the limits of optical resolution and the density of transcripts in single cells. Here we demonstrate an evolution of sequential fluorescence in situ hybridization (seqFISH+). We show that seqFISH+ can image mRNAs for 10,000 genes in single cells—with high accuracy and sub-diffraction-limit resolution—in the cortex, subventricular zone and olfactory bulb of mouse brain, using a standard confocal microscope. The transcriptome-level profiling of seqFISH+ allows unbiased identification of cell classes and their spatial organization in tissues. In addition, seqFISH+ reveals subcellular mRNA localization patterns in cells and ligand–receptor pairs across neighbouring cells. This technology demonstrates the ability to generate spatial cell atlases and to perform discovery-driven studies of biological processes in situ

Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior

The ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) contains ∼4,000 neurons that project to multiple targets and control innate social behaviors including aggression and mounting. However, the number of cell types in VMHvl and their relationship to connectivity and behavioral function are unknown. We performed single-cell RNA sequencing using two independent platforms—SMART-seq (∼4,500 neurons) and 10x (∼78,000 neurons)—and investigated correspondence between transcriptomic identity and axonal projections or behavioral activation, respectively. Canonical correlation analysis (CCA) identified 17 transcriptomic types (T-types), including several sexually dimorphic clusters, the majority of which were validated by seqFISH. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity

Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior

The ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) contains ∼4,000 neurons that project to multiple targets and control innate social behaviors including aggression and mounting. However, the number of cell types in VMHvl and their relationship to connectivity and behavioral function are unknown. We performed single-cell RNA sequencing using two independent platforms—SMART-seq (∼4,500 neurons) and 10x (∼78,000 neurons)—and investigated correspondence between transcriptomic identity and axonal projections or behavioral activation, respectively. Canonical correlation analysis (CCA) identified 17 transcriptomic types (T-types), including several sexually dimorphic clusters, the majority of which were validated by seqFISH. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity

Functional Characterization of the HopF Family of Type III Effectors in Arabidopsis thaliana

Pseudomonas syringae is a Gram-negative bacterial phytopathogen that causes disease in economically important crops. An essential virulence strategy of P. syringae involves injecting type III secretion effectors (T3SEs) into host cells to inhibit immune signaling. Advances in genomic sequencing have lead to an increase in the available number of sequences of plant bacterial pathogens, which has lead to identification of divergent homologs of known T3SEs. The HopF family of T3SE contains over 40 diverse family members. In order to explore the functional diversification of HopF family, this thesis undertakes the functional characterization of HopF T3SE in Arabidopsis thaliana. Functional analysis revealed multiple members with novel immune responses in Arabidopsis. A reverse genetics approach was used to identify the mechanism underlying the novel immune responses triggered by HopF family of T3SE. The Arabidopsis R protein X was required for recognition of a novel member of the HopF family.M.Sc

Method for High-Throughput, In Situ Characterization of AAV Variant Pools in Intact Tissue Using Ultrasensitive Sequential FISH

Extensive efforts have been made to engineer adeno-associated viruses (AAVs) with desirable characteristics, such as enhanced transduction efficiency and tissue- or cell-type specific tropisms. In-vivo selection, followed by next-generation sequencing (NGS)-based screening, has enabled us to uncover novel viral capsid variants, such as the AAV-PHP series (Deverman et al., Nat Biotech, 2016; Chan et al., Nat Neurosci, 2017; Kumar et al., Nat Methods, 2020). Despite successful library-based selections, the characterization of viral tropisms is slow and labor-intensive and is thus limited to only a handful of variants. To overcome this bottleneck and allow for high-throughput screening, we introduce an imaging-based approach that detects viral transcripts in intact tissue by using ultrasensitive, sequential fluorescence in situ hybridization (FISH). We first developed a new FISH method to enable detection of relatively low abundance viral transcripts compared to endogenous genes in tissue. Compared to two signal amplification methods, rolling-circle amplification (RCA) and hybridization chain reaction (HCR), our method resulted in a 2.7- or 6.7-fold higher signal-to-background ratio, respectively, with the same number of probes. The high sensitivity of our method also allowed us to detect RNA transcripts with 1 probe and distinguish capsid variants packaging an identical viral genome with a short mutated region (7 amino acids, equivalent to 21 base pairs) transduced in HEK293T cells. We also developed an efficient two-step probe stripping method to enable multiple rounds of labeling (up to 8), which increases the number of targets that can be characterized in the same tissue beyond the spectral limit (e.g., 4 colors x 8 rounds = 32 variants). The high sensitivity and ability for sequential labeling allowed us to examine the cell-type tropism of capsid variants and/or gene regulatory elements in intact tissue. For this purpose, we generated AAV pools, comprising a combination of novel AAV-PHP.B-like capsids and cell-type specific promoters, that package the same coding sequence with a unique barcode in the 3’UTR. The pool was injected into one animal at a low dose (~1e10 for each), and after 3-4 weeks of injection, the transcripts of each variant were detected with a custom probe set targeting the unique barcodes. As a proof-of-concept, we were able to characterize the cell-type tropism of 6 variants in one tissue within 4 hours. Further refinement of barcode designs (e.g., temporal barcoding or in situ sequencing) and single-molecule imaging will allow us to either reduce the screening time or increase the number of variants that can be characterized to hundreds. These approaches enable high-throughput characterization of virally delivered transgenes in intact tissue, thus complementing the active field of viral vector engineering with scalable tropism identification or validation. Moreover, visualizing the distribution of many variants while preserving spatial context will offer insights into AAV biology, which can include entry mechanisms as well as cell- and tissue-type associated expression