Selective Targeting of a Defined Subpopulation of Corticospinal Neurons using a Novel Klhl14-Cre Mouse Line Enables Molecular and Anatomical Investigations through Development into Maturity

Abstract

The corticospinal tract (CST) facilitates skilled, precise movements, which necessitates that subcerebral projection neurons (SCPN) establish segmentally specific connectivity with brainstem and spinal circuits. Developmental molecular delineation enables prospective identification of corticospinal neurons (CSN) projecting to thoraco-lumbar spinal segments; however, it remains unclear whether other SCPN subpopulations in developing sensorimotor cortex can be prospectively identified in this manner. Such molecular tools could enable investigations of SCPN circuitry with precision and specificity. During development, Kelch-like 14 (Klhl14) is specifically expressed by a specific SCPN subpopulation, CSNBC-lat, that reside in lateral sensorimotor cortex with axonal projections exclusively to bulbar-cervical targets. In this study, we generated Klhl14-T2A-Cre knock-in mice to investigate SCPN that are Klhl14+ during development into maturity. Using conditional anterograde and retrograde labeling, we find that Klhl14-Cre is specifically expressed by CSNBC-lat only at specific developmental time points. We establish conditional viral labeling in Klhl14-T2A-Cre mice as a new approach to reliably investigate CSNBC-lat axon targeting and confirm that this identifies known molecular regulators of CSN axon targeting. Therefore, Klhl14-T2A-Cre mice can be used as a novel tool for identifying molecular regulators of CST axon guidance in a relatively high-throughput manner in vivo. Finally, we demonstrate that intersectional viral labeling enables precise targeting of only Klhl14-Cre+ CSNBC-lat in the adult central nervous system. Together, our results establish that developmental molecular delineation of SCPN subpopulations can be used to selectively and specifically investigate their development, as well as anatomical and functional organization into maturity. Significance StatementThe cortex connects to brainstem and spinal targets through subcerebral projection neurons (SCPN), which exhibit molecular diversity during development based on their neocortical location and axonal targets. We generated a novel Klhl14-Cre mouse line to utilize this developmental delineation and drive Cre expression in a specific SCPN subpopulation. This developmental specificity enabled investigation of 1) areal locations of Klhl14+ SCPN in mature cortex, 2) their axonal collateralization at maturity, and 3) which genes can control their axon targeting. Using intersectional tools, we can also selectively label these neurons in the adult CNS. Therefore, developmental molecular delineation of SCPN not only provides prospective identification but also enables molecular analysis during development, as well as anatomical and functional investigations in adulthood.