Developmentally determined intersectional genetic strategies to dissect adult sensorimotor function

Abstract

SUMMARYImprovements in the speed and cost of expression profiling of neuronal tissues offer an unprecedented opportunity to define ever finer subgroups of neurons for functional studies. In the spinal cord, single cell RNA sequencing studies1,2support decades of work on spinal cord lineage studies3–5, offering a unique opportunity to probe adult function based on developmental lineage. While Cre/Flp recombinase intersectional strategies remain a powerful tool to manipulate spinal neurons6–8, the field lacks genetic tools and strategies to restrict manipulations to the adult mouse spinal cord at the speed at which new tools develop. This study establishes a new workflow for intersectional mouse-viral strategies to dissect adult spinal function based on developmental lineages in a modular fashion. To restrict manipulations to the spinal cord, we generate a brain-sparingHoxb8FlpOmouse line restricting Flp recombinase expression to caudal tissue. Recapitulating endogenousHoxb8gene expression9, Flp-dependent reporter expression is present in the caudal embryo starting day 9.5. This expression restricts Flp activity in the adult to the caudal brainstem and below.Hoxb8FlpOheterozygous and homozygous mice do not develop any of the sensory or locomotor phenotypes evident in Hoxb8 heterozygous or mutant animals10,11, suggesting normal developmental function of the Hoxb8 gene and protein inHoxb8FlpOmice. Compared to the variability of brain recombination in available caudal Cre and Flp lines12,13Hoxb8FlpOactivity is not present in the brain above the caudal brainstem, independent of mouse genetic background. Lastly, we combine theHoxb8FlpOmouse line with dorsal horn developmental lineage Cre mouse lines to express GFP in developmentally determined dorsal horn populations. Using GFP-dependent Cre recombinase viruses14and Cre recombinase-dependent inhibitory chemogenetics, we target developmentally defined lineages in the adult. We show how developmental knock-out versus transient adult silencing of the same RORβlineage neurons affects adult sensorimotor behavior. In summary, this new mouse line and viral approach provides a blueprint to dissect adult somatosensory circuit function using Cre/Flp genetic tools to target spinal cord interneurons based on genetic lineage.In briefWe describe the generation of aHoxb8FlpOmouse line that targets Flp-recombinase expression to the spinal cord, dorsal root ganglia, and caudal viscera. This line can be used in intersectional Cre/Flp strategies to restrict manipulations to the caudal nervous system. Additionally, we describe an intersectional genetics+viral strategy to convert developmental GFP expression into adult Cre expression, allowing for modular incorporation of viral tools into intersectional genetics. This approach allows for manipulation of a developmentally determined lineage in the adult. This strategy is also more accessible than traditional intersectional genetics, and can adapt to the constantly evolving available viral repertoire.Highlights-A newHoxb8FlpOmouse line allows Flp-dependent recombination in the spinal cord, dorsal root ganglia, and caudal viscera.-We observed no ectopic brain expression across mouse genetic backgrounds with theHoxb8FlpOmouse line.-Combining this new mouse line for intersectional genetics and a viral approach, we provide a novel pipeline to target and manipulate developmentally defined adult spinal circuits.

Human OPRM1 and murine Oprm1 promoter driven viral constructs for genetic access to μ-opioidergic cell types

“…This question of tropism and determination of the correct serotype to employ to best transduce a tissue or cell type is not unique to our viral constructs, however, as consensuses on the specific capsid proteins and construct elements that provide the greatest transduction efficacy within different tissue types and cell populations is still a matter of debate and investigation, requiring more meticulous documentation and data sharing amongst researchers across disciplines 88 . Examining the extent of the combinatorial approaches that can be taken with the use of our viruses with other transgenic animal lines and tools for the intersectional targeting of unique CNS and PNS cell populations 59 or the dissection of mu opioidergic circuits within the brain will thus require careful testing and planning when using our MORp constructs across any and all applications. Regardless, we believe that the results of these initial use case studies highlight the power and versatility of our MORp tools in the study of the opioidergic system not only in regards to the modulation of physiological pain responses, but also potentially in the study of chronic opioid addiction, withdrawal and other emerging complex behavioral features of pain and OUD as well.…”

“…16), such as the recently engineered PHP.eB and PNS PHP.S capsids variants, which are capable of selectively transducing cells within the CNS or the PNS over cells present in non-neural tissue types, respectively 12,57 , as well as the inclusion of specific promoter and/or enhancer element into constructs packaged within AAVs. Furthermore, hundreds of existing Cre and Flp recombinase transgenic mouse lines are also in use in labs around the world which can be used to achieve cell and circuit specific genetic access 58,59 . To capitalize on these advances in targeting strategies and tool development, we created four example mMORp constructs useful for intersectional neuronal labeling and tracing studies: a mMORp-mCherry-IRES-Cre construct encoding Cre recombinase, a mMORp-FlpO construct encoding Flipase, and two mMORp-eYFP constructs packaged in AAV-PHP.eB and AAV-PHP.s capsids.…”

Tetracycline transactivator overexpression in keratinocytes triggers a TRPV1 primary sensory neuron-dependent neuropathic itch

“…We also examined an intersectional TIGRE2.0 line, Ai195, in which CAG-tTA2 is activated after FLP-FRT recombination independent of the reporter gene, lox-STOP-lox-TRE-GCaMP7s (Supplemental Figure 3H). To execute conditional FLP-FRT recombination, we acquired the Hoxb8-FLPo line, in which FLPo recombinase is expressed in all tissues caudal to C7 (Bohic et al 2023) (Supplemental Figure 3I-J). Strikingly, double transgenic, Hoxb8-FLPo; Ai195 mice repeatedly licked/bit at their lower abdomen region of skin, inducing skin lesions that progressed in size over weeks, analogous to the scratching-induced lesions in the Phox2a; tTA-GCaMP6 mice (Supplemental Figure 3K).…”