Abstract
Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (optoα1AR) and cAMP (optoß2AR), or Ca2+-permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in Hcn4BAC-CatCh2/Myh6-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.
Highlights
Optogenetic effectors and sensors enable the interrogation of complex biological signaling networks at the molecular level in vivo (Akerboom et al, 2013; Fenno et al, 2011; Gengyo-Ando et al, 2017; Kotlikoff, 2007; Tsien, 2003)
The optogenetic effector proteins we selected for this toolbox were optoα1AR (Airan et al, 2009), optoß2AR (Airan et al, 2009), and CatCh2 (Zhang et al, 2006) for light activation of InsP3 or cAMP secondary messenger pathways or Ca2+ membrane channel, respectively
Each of the open reading frames for the optogenetic proteins were linked to an internal ribosome entry sequence (IRES) element and bacterial LacZ ORF for simple screening
Summary
Optogenetic effectors and sensors enable the interrogation of complex biological signaling networks at the molecular level in vivo (Akerboom et al, 2013; Fenno et al, 2011; Gengyo-Ando et al, 2017; Kotlikoff, 2007; Tsien, 2003) The power of these molecular tools is optimized in mammalian systems by genetic lineage specification and combinatorial strategies. Cell Biology specificity by crossbreeding Cre recombinase and floxed responder mouse lines (Zariwala et al, 2012), the attendant allelic requirements render this approach impractical for combinatorial experiments using multiple optogenetic tools Such experiments are facilitated by the design and assembly of sets of individual monoallelic lines that strategically place spectrally distinct optogenetic effectors and sensors in interacting cell lineages, allowing simple crosses to probe cell-cell signaling. Interacting vascular, neural, and muscular tissues are targeted through a variety of genetic strategies, expression specificity confirmed, and examples of their use provided
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