Genetic dissection of the glutamatergic neuron system in cerebral cortex

Article Genetic dissection of the glutamatergic neuron system in cerebral cortex https://doi.org/10.1038/s41586-021-03955-9 Received: 23 April 2020 Accepted: 25 August 2021 Published online: 6 October 2021 Katherine S. Matho1, Dhananjay Huilgol1,2,11, William Galbavy1,3,11, Miao He1,8,11, Gukhan Kim1,11, Xu An1,2, Jiangteng Lu1,9, Priscilla Wu1, Daniela J. Di Bella4, Ashwin S. Shetty4, Ramesh Palaniswamy1, Joshua Hatfield1,2, Ricardo Raudales1,3, Arun Narasimhan1, Eric Gamache1, Jesse M. Levine1,5, Jason Tucciarone1,5,10, Eric Szelenyi1, Julie A. Harris5,6, Partha P. Mitra1, Pavel Osten1, Paola Arlotta4,7 & Z. Josh Huang1,2 ✉ Open access Check for updates Diverse types of glutamatergic pyramidal neurons mediate the myriad processing streams and output channels of the cerebral cortex1,2, yet all derive from neural progenitors of the embryonic dorsal telencephalon3,4. Here we establish genetic strategies and tools for dissecting and fate-mapping subpopulations of pyramidal neurons on the basis of their developmental and molecular programs. We leverage key transcription factors and effector genes to systematically target temporal patterning programs in progenitors and differentiation programs in postmitotic neurons. We generated over a dozen temporally inducible mouse Cre and Flp knock-in driver lines to enable the combinatorial targeting of major progenitor types and projection classes. Combinatorial strategies confer viral access to subsets of pyramidal neurons defined by developmental origin, marker expression, anatomical location and projection targets. These strategies establish an experimental framework for understanding the hierarchical organization and developmental trajectory of subpopulations of pyramidal neurons that assemble cortical processing networks and output channels. Pyramidal neurons (PyNs) constitute the large majority of nerve cells in the cerebral cortex and mediate all of the inter-areal processing streams and output channels1,2,4. Traditionally, PyNs have been classified into several major classes according to their laminar location and broad axon projection targets, such as intratelencephalic (IT) and extratelencephalic (ET or corticofugal), which further comprises subcerebral (including pyramidal tract; PT) and corticothalamic (CT) PyNs1. Within these classes, subsets of PyNs form specific local and long-range connectivity, linking discrete microcircuits to cortical subnetworks and output channels1,5. Single-cell transcriptome analysis suggests that there are over fifty PyN transcriptomic types6. However, genetic tools and strategies for experimentally accessing PyN subpopulations are limited. All PyNs are generated from neural progenitors in the embryonic dorsal telencephalon, where regionally differentiated radial glial progenitors (RGs) undergo asymmetric divisions, giving rise to radial clones of PyNs that migrate to the cortex in an inside-out order7. RGs generate PyNs either directly or indirectly through intermediate progenitors (IPs), which divide symmetrically to generate pairs of PyNs8. A set of temporal patterning genes drive lineage progression in RGs, which unfold a conserved differentiation program in successively generated postmitotic neurons3,4,9. Resolving the lineage organization of diverse progenitors and their relationship to projection-defined PyN subpopulations requires fate-mapping tools with cell type and temporal resolution2. Here we present strategies and a genetic toolkit in the mouse for targeting PyN subpopulations and progenitors guided by knowledge of their developmental programs. We leverage gene expression patterns of the cell-type specification and differentiation programs to target biologically significant progenitor subsets, PyN subpopulations and their developmental trajectories (Fig. 1a–c, Extended Data Table 1). These tools and strategies provide a roadmap for accessing hierarchically organized PyN types at progressively finer resolution. They will facilitate the tracking of developmental trajectories of PyNs for elucidating the organization and assembly of neural circuits of the cerebral hemisphere, including the cortex, hippocampus and basolateral amygdala. Fate-mapping PyN progenitors RGs The transcription factors LHX2 and FEZF2 act at multiple stages throughout corticogenesis10–12. The fate potential of and relationship between Lhx2+ RGs (RGsLhx2) and Fezf2+ RGs (RGsFezf2) are largely unknown. We generated Lhx2-CreER, Fezf2-CreER and Fezf2-Flp driver lines and performed a series of fate-mapping experiments at multiple 1 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY, USA. 2Department of Neurobiology, Duke University Medical Center, Durham, NC, USA. 3Program in Neuroscience, Department of Neurobiology and Behavior, Stony Brook University, Stony Brook, NY, USA. 4Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA. 5 Program in Neuroscience and Medical Scientist Training Program, Stony Brook University, New York, NY, USA. 6Allen Institute for Brain Science, Seattle, WA, USA. 7Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA. 8Present address: Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China. 9Present address: Shanghai Jiaotong University Medical School, Shanghai, China. 10Present address: Department of Psychiatry, Stanford University School of Medicine, Palo Alto, CA, USA. 11These authors contributed equally: Dhananjay Huilgol, William Galbavy, Miao He, Gukhan Kim. ✉e-mail: 182 | Nature | Vol 598 | 7 October 2021 SC Pn PT CT Spd b SVZ VZ RG Lhx2 c Tis21 Tbr2 Foxp2 Tbr1 Tle4 Fezf2 Sema3e Adcyap1 Tcerg1l Lhx2 Cux1 Plxnd1 Tbr2 Fezf2 Progenitor temporal patterning IT Tbr1 Foxp2 Tle4 Fezf2 Adcyap1 Tcerg1l Sema3e Lhx2 L4 Cux1 Tbr2 L5 >E15 Plxnd1 L6 Neuronal differentiation g Fezf2 L2 L3 Cortex/ Thalamus Subcerebral targets Striatum Postmitotic restriction Lhx2 L1 L2 f L3 L3 L4 L4 L5a L5a Hem L5b L5b Hem L6 Lhx2-CreER;Ai14 E12.5→P30 E12.5→E13.5 Lhx2-CreER IS STOP RFP GFP Lhx2+Fezf2– Lhx2+Fezf2+ E12.5→P30 E12.5→E13.5 E12.5→E13.5 Hem ## # # D M * 0 ** † † † # L * * * 5 10 15 20 25 PyN (SSp-bfd) (%) m m n 0 600 n * * 400 0 100 0 Lhx2+ Fezf2+ * * 500 200 400 300 200 L2 L3 L4 L5a L5b L6 l Lhx2+Fezf2– Lhx2+Fezf2+ 1,000 Fezf2 Lhx2 Fezf2-CreER;Ai14 1,500 Fezf2-Flp Fezf2 Lhx2 Lhx2 L6 k j i h L2 No. of progenitors Str Th e No. of progenitors d a † # † # † R M C E12.5→P30 Fig. 1 | Strategies and drivers to target PyN types and fate-map progenitors. a, Major PyN projection classes mediating intratelencephalic streams (IT, red) and cortical output channels (PT, blue; CT, purple) in a sagittal brain section. Pn, pons; SC, superior colliculus; Str, striatum; Th, thalamus; Spd, spinal cord. b, PyN developmental trajectory. R (...truncated)