Targeting Tmem63b and Piezo2 in C-fiber low threshold mechanoreceptor: limitation of Vglut3-IRES-Cre

Abstract

Peripheral somatosensory neurons in the dorsal root ganglia (DRG) transduce mechanical force in the skin and other organs into electrical signals using specialized mechanically activated (MA) ion channels that initiate neuronal activation in response to force. Increasing evidence highlights PIEZO2 as the primary transducer of low-threshold mechanical force in DRG neurons. However, in the absence of Piezo2, mice and humans still respond to noxious painful stimuli like pinch, suggesting that additional MA channel(s) likely exist in DRG neurons. Strategies to identify Cre lines and DRG subpopulations that select for non-PIEZO2 expressing neurons is therefore an ongoing effort in the field to discover unknown mechanosensors. Here, we investigated a Vglut3 labeled mouse line as a candidate to identify non-PIEZO2 MA channels in a subtype of DRG neurons called C-fiber low threshold mechanoreceptors (C-LTMRs). Our study carefully demonstrates that the Vglut3-IRES-Cre mouse line specifically and efficiently labels C-LTMR neurons of the DRG. Electrophysiological recordings using two different in vitro mechanical stimulation assays show that the genetically labelled Vglut3 neurons have robust indentation- and stretch-activated MA currents that are exclusively slowly or ultra-slowly adapting. To determine whether the Vglut3-IRES-Cre mouse line can be used to delete genes of interest and identify the underlying MA ion channels in C-LTMRs we attempted to generate a Tmem63b conditional knockout using this Cre-line but detected incomplete loss of Tmem63b transcript and lack of TMEM63B- dependent effect on C-LTMR MA currents. Together, our results emphasize that although the Vglut3-IRES-Cre line is robust in driving expression of a conditional reporter gene, it is inefficient in deleting genes like Tmem63b as well as Piezo2. Statement of SignificanceIn this study, we identify a previously uncharacterized Vglut3-IRES-Cre as a tool to uncover unknown mechanosensors in C-LTMRs of the DRG. We methodically demonstrate that the Vglut3-IRES-Cre line labels C-LTMRs and express two mechanosensors, Tmem63b and Piezo2. However, we were unable to discern which gene contributes to the MA currents due to poor knockout efficiency induced by the Vglut3-IRES-Cre. Our studies highlight the nuances and challenges associated with Cre lines and alert future investigations to exercise caution while using Cre lines to discover the contribution of specific genes to mechanosensation.