Voltage-Seq: all-optical postsynaptic connectome-guided single-cell transcriptomics.

Understanding the routing of neuronal information requires the functional characterization of connections. Neuronal projections recruit large postsynaptic ensembles with distinct postsynaptic response types (PRTs). PRT is typically probed by low-throughput whole-cell electrophysiology and is not a selection criterion for single-cell RNA-sequencing (scRNA-seq). To overcome these limitations and target neurons based on specific PRTs for soma harvesting and subsequent scRNA-seq, we created Voltage-Seq. We established all-optical voltage imaging and recorded the PRT of 8,347 neurons in the mouse periaqueductal gray (PAG) evoked by the optogenetic activation of ventromedial hypothalamic (VMH) terminals. PRTs were classified and spatially resolved in the entire VMH-PAG connectome. We built an onsite analysis tool named VoltView to navigate soma harvesting towards target PRTs guided by a classifier that used the VMH-PAG connectome database as a reference. We demonstrated Voltage-seq by locating VMH-driven γ-aminobutyric acid-ergic neurons in the PAG, guided solely by the onsite classification in VoltView.

All-optical voltage imaging-guided postsynaptic single-cell transcriptome profiling with Voltage-Seq.

Neuronal pathways recruit large postsynaptic populations and maintain connections via distinct postsynaptic response types (PRTs). Until recently, PRTs were accessible as a selection criterion for single-cell RNA sequencing only through probing by low-throughput whole-cell electrophysiology. To overcome these limitations and target neurons on the basis of specific PRTs for soma collection and subsequent single-cell RNA sequencing, we developed Voltage-Seq using the genetically encoded voltage indicator Voltron in acute brain slices from mice. We also created an onsite analysis tool, VoltView, to guide soma collection of specific PRTs using a classifier based on a previously acquired database of connectomes from multiple animals. Here we present our procedure for preparing the optical path, the imaging setup and detailing the imaging and analysis steps, as well as a complete procedure for sequencing library preparation. This enables researchers to conduct our high-throughput all-optical synaptic assay and to obtain single-cell transcriptomic data from selected postsynaptic neurons. This also allows researchers to resolve the connectivity ratio of a specific pathway and explore the diversity of PRTs within that connectome. Furthermore, combining high throughput with quick analysis gives unique access to find specific connections within a large postsynaptic connectome. Voltage-Seq also allows the investigation of correlations between connectivity and gene expression changes in a postsynaptic cell-type-specific manner for both excitatory and inhibitory connections. The Voltage-Seq workflow can be completed in ~6 weeks, including 4-5 weeks for viral expression of the Voltron sensor. The technique requires knowledge of basic laboratory techniques, micromanipulator handling skills and experience in molecular biology and bioinformatics.

Elastic dosage compensation by X-chromosome upregulation.

X-chromosome inactivation and X-upregulation are the fundamental modes of chromosome-wide gene regulation that collectively achieve dosage compensation in mammals, but the regulatory link between the two remains elusive and the X-upregulation dynamics are unknown. Here, we use allele-resolved single-cell RNA-seq combined with chromatin accessibility profiling and finely dissect their separate effects on RNA levels during mouse development. Surprisingly, we uncover that X-upregulation elastically tunes expression dosage in a sex- and lineage-specific manner, and moreover along varying degrees of X-inactivation progression. Male blastomeres achieve X-upregulation upon zygotic genome activation while females experience two distinct waves of upregulation, upon imprinted and random X-inactivation; and ablation of Xist impedes female X-upregulation. Female cells carrying two active X chromosomes lack upregulation, yet their collective RNA output exceeds that of a single hyperactive allele. Importantly, this conflicts the conventional dosage compensation model in which naïve female cells are initially subject to biallelic X-upregulation followed by X-inactivation of one allele to correct the X dosage. Together, our study provides key insights to the chain of events of dosage compensation, explaining how transcript copy numbers can remain remarkably stable across developmental windows wherein severe dose imbalance would otherwise be experienced by the cell.

Frequent and biased odorant receptor (OR) re-selection in an olfactory placode-derived cell line.

We previously characterized a clonal olfactory placode-derived cell line (OP6) as a model system for studying odorant receptor (OR) choice, where individual OP6 cells, similar to olfactory sensory neurons in vivo, transcribe one allele ("monoallelic") of one OR gene ("monogenic"). The OP6 cell line provides a unique opportunity to investigate intrinsic properties of OR regulation that cannot easily be investigated in vivo. First, whereas OR-expressing cells in vivo are post-mitotic, OP6 cells are immortalized, raising interesting questions about the stability of epigenetic states associated with OR selection/silencing as OP6 cells progress through the cell cycle. Second, OP6 cells have been isolated away from extrinsic developmental cues, and therefore, any long-term OR selection biases are likely to arise from intrinsic epigenetic states that persist in the absence of developmental context. In this study, we investigated OR re-selection frequency and selection biases within clonal OP6 cell populations. We found no evidence of OR stability through the cell cycle: our results were most consistent with OR re-selection events transpiring at least once per cell division, suggesting that chromatin states associated with OR selection in this system might not be maintained in the subsequent generation. In contrast, we found strong evidence for OR selection biases maintained over prolonged culturing across a diverse set of OP6 cell lineages, suggesting the persistence of intrinsic epigenetic states that advantage some OR loci over others. Together, our data suggest that in the absence of instructive cues, intrinsic epigenetic states influencing OR eligibility, but not those determining OR choice, might persist through the cell cycle.

Factor interactions with the simian virus 40 early pre-mRNA influence branch site selection and alternative splicing.

To study the interaction of splicing factors with the simian virus 40 early-region pre-RNA, which can be alternatively spliced to produce large T and small t mRNAs, we used an in vitro RNase protection assay that defines the 5' boundaries of factor-RNA interactions. Protection products reflecting factor interactions with the large T and small t 5' splice sites and with the multiple lariat branch site region were characterized. All protection products were detected very early in the splicing reaction, before the appearance of spliced RNAs. However, protection of the large T 5' splice site was detected well before small t 5' splice site and branch site protection products, which appeared simultaneously. Oligonucleotide-targeted degradation of small nuclear RNAs (snRNAs) revealed that protection of the branch site region, which occurred at multiple sites, required intact U2 snRNA and was enhanced by U1 snRNA, while protection of the large T and small t 5' splice sites required both U1 and U2 snRNAs. Analysis of several pre-RNAs containing mutations in the branch site region suggests that factor interactions involving the multiple copies of the branch site consensus determine the selection of branch points, which is an important factor in the selection of alternative splicing pathways.

Alternative splicing of SV40 early pre-mRNA is determined by branch site selection.

Splicing of SV40 early pre-mRNA to alternative large-T and small-t mRNAs involves the utilization of multiple lariat branch sites. To determine the functional significance of these sites, we constructed and analyzed a set of base substitution mutants in which the major branch acceptors were altered, either singly or in combination. The ratio of large-T to small-t mRNAs produced in vivo was found to vary by over 100-fold between different mutants. When splicing was assayed in vitro with a standard pre-RNA, which results in splicing almost exclusively to large-T mRNA, the patterns of branch site utilization were altered dramatically, although the mutations were essentially without effect on splicing efficiency. However, use of a 5' truncated pre-RNA, which results in a splicing pattern that reflects the in vivo alternative splicing potential of this pre-RNA, revealed a strong correlation between the effects of the base substitutions on branch site selection and alternative splice-site utilization. An RNase protection analysis of factor interactions with the 5' splice sites and branch sites in wild-type and mutant pre-RNAs suggests that a competition for different branch sites plays a crucial role in the assembly of 'alternative' spliceosomes, thereby controlling alternative splice-site selection.

In vitro splicing of simian virus 40 early pre mRNA.

The products of splicing of simian virus 40 early pre mRNA in HeLa cell nuclear extracts have been characterized. Of the two alternative splicing patterns exhibited by this precursor in vivo, which involve the use of alternative large T and small t 5' splice sites and a single shared 3' splice site, only one, producing large T mRNA, was found to occur in vitro. A number of possible intermediates and byproducts of splicing of large T mRNA were observed, including free large T 5' exon, lariat form intron joined to 3' exon and free lariat and linear forms of large T intron. The formation of these products argues strongly for a basic similarity in the mechanism underlying large T and other, non-alternative splices. A collection of RNAs resulting from protection of early pre mRNA at specific points from an endogenous 5' to 3' exonuclease activity in vitro have also been observed. The regions of the precursor RNA protected map to positions immediately upstream of the 5' splice sites of large T and small t and the lariat branchpoint, and may represent interaction of these regions with components of the splicing machinery.

Splicing of SV40 early pre-mRNA to large T and small t mRNAs utilizes different patterns of lariat branch sites.

To explore the mechanism and control of alternative splicing, we have characterized the products formed by splicing of SV40 early pre-mRNA in vitro and in vivo. Large T and small t mRNAs are derived from this precursor by joining alternative 5' splice sites to a single shared 3' splice site. In contrast to pre-mRNAs studied previously, we have shown that splicing to large T RNA involves the utilization of multiple lariat branch sites, while small t splicing uses a single branch site. Interestingly, the predominant branch sites utilized in splicing of large T RNA in vitro were found to differ in nuclear extracts from HeLa and human 293 cells, correlated with previously observed differences in the ratio of large T to small t mRNAs produced in the two cell types. To test the significance of this correlation, we examined the products formed by splicing of an SV40 early precursor microinjected into X. laevis oocytes. Strikingly, both the pattern of branch sites used in large T splicing and the ratio of large T to small t mRNAs produced were found to be identical to those observed in 293 cells and extracts.