bioRxiv preprint doi: https://doi.org/10.1101/409458; this version posted September 6, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Single-cell transcriptional dynamics and origins of neuronal diversity in the developing mouse neocortex L. Telley1,3*†, G. Agirman1,4†, J. Prados1, S. Fièvre1, P. Oberst1, I. Vitali1, L. Nguyen4, A. Dayer1,5, D. Jabaudon1,2* 1 Department of Basic Neurosciences, University of Geneva, Geneva, Switzerland. 2 Clinic of Neurology, Geneva University Hospital, Geneva, Switzerland. 3 Current address: Department of Basic Neuroscience, University of Lausanne, Switzerland. 4 GIGA-Neurosciences, University of Liège, C.H.U. Sart Tilman, Liège, Belgium. 5 Department of Psychiatry, Geneva University Hospital, Geneva, Switzerland. † equally contributed to this work. * Correspondence to: [email protected]; [email protected] During cortical development, distinct subtypes of glutamatergic neurons are sequentially born and differentiate from dynamic populations of progenitors. The neurogenic competence of these progenitors progresses as corticogenesis proceeds; likewise, newborn neurons transit through sequential states as they differentiate. Here, we trace the developmental transcriptional trajectories of successive generations of apical progenitors (APs) and isochronic cohorts of their daughter neurons using parallel single-cell RNA sequencing between embryonic day (E) 12 and E15 in the mouse cerebral cortex. Our results identify the birthdate- and differentiation stage- related transcriptional dynamics at play during corticogenesis. As corticogenesis proceeds, APs transit through embryonic age-dependent molecular states, which are transmitted to their progeny to generate successive initial daughter cell identities. In neurons, essentially conserved post-mitotic differentiation programs are applied onto these distinct AP-derived ground states, allowing temporally-regulated sequential emergence of specialized neuronal cell types. Molecular temporal patterning of sequentially-born daughter neurons by their respective mother cell thus underlies emergence of neuronal diversity in the neocortex. One Sentence Summary: During corticogenesis, temporally dynamic molecular birthmarks are transmitted from progenitors to their post-mitotic progeny to generate neuronal diversity. The cerebral cortex is a cellularly heterogeneous 2016; Gao et al. 2014, Guo et al. 2013; Gaspard structure, whose neuronal circuits underlie high- et al. 2007); likewise, newborn neurons transit order cognitive and sensorimotor information through sequential transcriptional states as they processing. During embryogenesis, distinct differentiate (Zahr et al. 2018; Telley et al. 2016; subtypes of glutamatergic neurons are Azim et al. 2009). Although the single-cell sequentially born and differentiate from transcriptional diversity of the neocortex is populations of progenitors located in the increasingly well characterized (Saunders et al. germinal zones below the cortex (Jabaudon 2018; Zeisel et al. 2018; Kageyama et al. 2018; 2017; Florio & Huttner 2014). The aggregate Nowakowski et al. 2017; Tasic et al. 2016; neurogenic competence of ventricular zone Zeisel et al. 2015), little is yet known about the progenitors (i.e. apical progenitors, APs) molecular processes driving either the progresses as corticogenesis proceeds progression of AP competence, or the specific (Govindan & Jabaudon 2017; Okamoto et al. -1- bioRxiv preprint doi: https://doi.org/10.1101/409458; this version posted September 6, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. differentiation of daughter neurons born from axis, corresponding to the birth and maturation these progenitors at sequential embryonic ages. of daughter neurons, and a birthdate axis, corresponding to the temporal progression in To address these questions, we used AP transcriptional states at sequential FlashTag (FT), a high temporal resolution embryonic ages. These two cardinal processes method to pulse-label APs and their daughter are the major source of transcriptional diversity neurons (Telley et al. 2016; Govindan et al. in the developing neocortex. 2018), to trace the transcriptional trajectories of We used a graph-based cluster analysis successive generations of APs and isochronic to investigate the diversity of differentiation cohorts of their daughter neurons between stage- and birthdate-specific cells and identified embryonic day (E) 12 and E15. This four embryonic age-defined AP transcriptional corresponds to the period during which APs states, as well as two embryonic age-defined successively generate layer (L) 6, L5, L4 and basal progenitor populations, as recently L2/3 neurons (Jabaudon 2017). Following reported (Yuzwa et al. 2017) (Fig. 1C). Two microdissection of the putative somatosensory + classes of 1-day-old neurons (N1d) could be cortex, we collected FT cells by FACS after distinguished, early-born cells (i.e. E12-E13- either 1 h, as APs are still dividing, 24 h, as born) and later-born cells (i.e. E14-E15-born). daughter cells are transiting through the These two classes of neurons displayed early subventricular zone, or 96 h (i.e. four days), onset expression of deep- and superficial-layer once daughter neurons have entered the cortical markers, which foreshadowed their upcoming plate (Fig. 1A and fig. S1, A and B) (Telley et lamina-related identity (Fig. 1C). Classical al. 2016; Govindan et al. 2018). We performed deep-layer markers were also expressed by late- single-cell RNA sequencing at each of these 3 born neurons (fig S2A), consistent with an differentiation stages and 4 embryonic ages initial period of mixed identity followed by (E12, E13, E14, and E15), which yielded a total molecular cross-interactions and progressive of 2,756 quality-controlled cells across 12 fate refinement (Zahr et al. 2018; Ozair et al. conditions for analysis (fig. S1, C and D, and 2018; Azim et al. 2009). Accordingly, by four Methods). days of age, neurons with mutually-exclusive Analysis of cellular transcriptional expression of classical lamina-specific markers identities using t-SNE dimensionality reduction such as Bcl11b (an L5 marker), Rorb (L4) and revealed two main axes of organization: a Cux1 (L2/3) emerged. Of note, GABAergic differentiation (i.e. collection time) axis and a interneurons were also identified (Fig. 1C and birthdate (i.e. injection day) axis (Fig. 1B). fig. S2B), likely corresponding to cells Along the differentiation axis (Fig. 1B, left), 1 migrating into the dorsal pallium after FT h-, 1-day- and 4-day-old cells were organized labeling of their progenitors in the ventral into clusters which corresponded to (1) APs, (2) pallium (Govindan et al. 2018; Wamsley & basal progenitors (BPs) and 1-day-old neurons Fishell 2017; Marin 2013). Astrocytes, (N1d), and (3) 4-day-old neurons (N4d), as corresponding to 4-day-old daughter cells of indicated by the combined expression of type- E15 APs (Minocha et al. 2017; Cahoy et al. specific markers (Telley et al. 2016). Cells born 2008) were also present (Fig. 1C and fig. S2C). at successive times of corticogenesis were These two cell-types were not further organized perpendicularly to this differentiation investigated in this study. Differential axis, forming a birthdate axis (Fig. 1B, right). expression analysis identified type-enriched This chronotopic map was particularly apparent transcripts (Fig. 1D) whose temporal patterns of for APs and 1-day-old daughter cells, but less expression were confirmed using in situ striking in 4-day-old neurons, suggesting that hybridization (Fig. 1E; figs. S3 and S4; ISH; the salience of birthdate-related transcriptional Allen Developing Mouse Brain Atlas). features decreases with differentiation. Together, these results indicate that APs transit Together, these data reveal two orthogonal axes through temporally dynamic transcriptional of transcriptional organization: a differentiation states during corticogenesis as daughter neurons -2- bioRxiv preprint doi: https://doi.org/10.1101/409458; this version posted September 6, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Differentiation Birthdate Collection time Neurod2 Injection day +96h N ABFT 1h E12 +96h 24h Tubb3 E13 96h N4d E14 E15 Eomes N1d BP CP Btg2 +24h BP +24h AP Sox2 FT +1h AP Collection time SVZ Pax6 +1h VZ FT t-SNE2 E12 E13 E14 E15 E12 100 μm t-SNE1 Expression: Min Max Injection day Layer or cell-type C E12 1314 15 marker genes n cells D Enriched genes E E11.5E13.5 E15.5 early 190 mid1 228 early CP E15.5 P4 AP mid2 195 late 230 AP L2/3 1 VZ ly IN r L4 a e N4d early 107 n = 13 ISH L5 BP late 121 CP L6 N4d early 320 mid N1d N1d late 349 n = 13 AP early1 31 E18.5 P4 BP Astro early2 327 n = 6 N4d late1 146 2 L2/3 late2 205 L4 e AP lat L5 Astro 64 late N4d N4d L6 IN.1 142 AP Others IN.2 101 1 150 n = 8 n = 14 200 μm Rorb 0 100 Gfap Cux1 Sox5 Sox2 Gad2 Fezf2 Satb2 Expression (% max): 0 100 Expression: Min Max Bcl11b % total Eomes Neurod2 DL SL Fig. 1. Birthdate and differentiation stage-related cellular diversity in the developing neocortex. (A) Schematic illustration of the experimental procedure. M-phase APs were labeled by FT injection performed at either E12, E13, E14 or E15 and isochronic cohorts of APs and daughter cells were collected either 1, 24 or 96 hours later. (B) t-SNE representation of the single cell RNA sequencing dataset revealing the transcriptional organization of the cells according to the time at which they were collected (i.e. their differentiation status) and the day on which the injection was performed (i.e. their birthdate). APs, BPs and Ns can be distinguished by their combinatorial expression of key marker genes (n = 20 transcripts). (C) Cluster analysis reveals transcriptomically distinct and temporally dynamic cellular clusters. Cluster nomenclature reflects prevalence of the cluster at a given embryonic age (early: E12/E13, late: E14/E15).