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Methodology and theoretical basis of forward genetic screening for sleep/wakefulness in mice.

ABSTRACT: The regulatory network of genes and molecules in sleep/wakefulness remains to be elucidated. Here we describe the methodology and workflow of the dominant screening of randomly mutagenized mice and discuss theoretical basis of forward genetics research for sleep in mice. Our high-throughput screening employs electroencephalogram (EEG) and electromyogram (EMG) to stage vigilance states into a wake, rapid eye movement sleep (REMS) and non-REM sleep (NREMS). Based on their near-identical sleep/wake behavior, C57BL/6J (B6J) and C57BL/6N (B6N) are chosen as mutagenized and counter strains, respectively. The total time spent in the wake and NREMS, as well as the REMS episode duration, shows sufficient reproducibility with small coefficients of variance, indicating that these parameters are most suitable for quantitative phenotype-driven screening. Coarse linkage analysis of the quantitative trait, combined with whole-exome sequencing, can identify the gene mutation associated with sleep abnormality. Our simulations calculate the achievable LOD score as a function of the phenotype strength and the numbers of mice examined. A pedigree showing a mild decrease in total wake time resulting from a heterozygous point mutation in the Cacna1a gene is described as an example.

SUBMITTER: Miyoshi C

PROVIDER: S-EPMC6689935 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:SPEACC-seq is a novel high-throughput method which enables forward genetic screens to identify cell-cell interaction mechanisms that uncovered an astrocyte-microglia regulatory circuit mediated by amphiregulin and IL33-ST2. signaling. Cell-cell interactions in the central nervous system (CNS) play central roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. For example, several factors mediate microglia-astrocyte interactions that promote CNS pathology, but less is known about regulatory interactions that limit tissue pathology. Here we report the development of SPEACC-seq (Stimulation, Perturbation, and Encapsulation of interACting Cells followed by Sequencing), a forward genetic screening platform which combines genome-wide CRISPR/Cas9 perturbations, cell co-culture in picoliter droplets, and microfluidic-based fluorescence activated droplet sorting to identify mechanisms of cell-cell communication. Using SPEACC-seq in combination with an in vivo perturb-seq screen, we identified microglia-produced amphiregulin as a suppressor of disease promoting astrocyte responses in experimental autoimmune encephalomyelitis (EAE), a pre-clinical model of multiple sclerosis (MS). The production of microglial amphiregulin was induced via ST2 signaling by IL-33 released from astrocytes during EAE. Indeed, the genetic inactivation of ST2 or amphiregulin in microglia, or IL-33 or amphiregulin signaling in astrocytes resulted in the worsening of EAE, suggesting that IL-33-induced microglial amphiregulin limits disease-promoting astrocyte responses associated with CNS pathology. This regulatory loop was also detected in human astrocytes and microglia both in vitro and in MS patient CNS samples. In summary, we developed SPEACC-seq, a high-throughput, droplet-based forward genetic screening platform for the identification of cell-cell interaction mechanisms, which identified a novel microglia-astrocyte negative feedback loop that limits CNS pathology.

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Methodology and theoretical basis of forward genetic screening for sleep/wakefulness in mice.

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