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.
Postsynaptic plasticity is not accessible as a selection criterion for molecular identification of neurons by single-cell RNA-sequencing (scRNA-seq). The currently available methods to find specific connection plasticity ex vivo have inherently low throughput. To overcome these limitations and pre-select neurons based on short-term postsynaptic plasticity for soma harvesting and subsequent scRNA-seq we created Voltage-Seq. First, we established all-optical voltage imaging and recorded the short-term postsynaptic plasticity of 6911 periaqueductal gray (PAG) neurons evoked by optogenetic activation of the ventromedial hypothalamic (VMH) input. Postsynaptic response-types were classified and spatially resolved in the entire innervated PAG. Next, to browse and identify all-optical responses, we built a quick on-site analysis named VoltView which incorporated the a priori VMH-PAG connectome database as a classifier. VoltView targetedly identifies postsynaptic neurons for somatic harvesting. We demonstrated the agility of Voltage-Seq in locating GABAergic PAG neurons, guided by an all-optical connectivity map and on-site classification in VoltView.
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