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Temporal controls over inter-areal cortical projection neuron fate diversity

Abstract

Interconnectivity between neocortical areas is critical for sensory integration and sensorimotor transformations1,2,3,4,5,6. These functions are mediated by heterogeneous inter-areal cortical projection neurons (ICPN), which send axon branches across cortical areas as well as to subcortical targets7,8,9. Although ICPN are anatomically diverse10,11,12,13,14, they are molecularly homogeneous15, and how the diversity of their anatomical and functional features emerge during development remains largely unknown. Here we address this question by linking the connectome and transcriptome in developing single ICPN of the mouse neocortex using a combination of multiplexed analysis of projections by sequencing16,17 (MAPseq, to identify single-neuron axonal projections) and single-cell RNA sequencing (to identify corresponding gene expression). Focusing on neurons of the primary somatosensory cortex (S1), we reveal a protracted unfolding of the molecular and functional differentiation of motor cortex-projecting (\(\vec{{\rm{M}}}\)) ICPN compared with secondary somatosensory cortex-projecting (\(\vec{{\rm{S}}2}\)) ICPN. We identify SOX11 as a temporally differentially expressed transcription factor in \(\vec{{\rm{M}}}\) versus \(\vec{{\rm{S}}2}\) ICPN. Postnatal manipulation of SOX11 expression in S1 impaired sensorimotor connectivity and disrupted selective exploratory behaviours in mice. Together, our results reveal that within a single cortical area, different subtypes of ICPN have distinct postnatal paces of molecular differentiation, which are subsequently reflected in distinct circuit connectivities and functions. Dynamic differences in the expression levels of a largely generic set of genes, rather than fundamental differences in the identity of developmental genetic programs, may thus account for the emergence of intra-type diversity in cortical neurons.

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Fig. 1: Postnatal emergence of intracortical connections from S1.
Fig. 2: \(\overrightarrow{{\bf{M}}}\) and \(\overrightarrow{{\bf{S}}{\bf{2}}}\) ICPN have distinct maturation paces.
Fig. 3: Postnatal SOX11 expression levels regulate S1 sensorimotor connectivity.
Fig. 4: Subtype-specific ICPN connectivity underlies specific features of sensorimotor exploration.

Data availability

scRNA-seq data have been deposited in the Gene Expression Omnibus (GEO) under the following accessions: MAPseq, GSE118681; Retrobeads: GSE116944; ConnectID: GSE156080.

Code availability

https://github.com/pradosj/docker_sindbis

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Acknowledgements

We thank A. Zador for his contribution to an earlier version of this manuscript and for his sharing of MAPseq mapping reagents; the Genomics Platform and FACS Facility of the University of Geneva; L. Frangeul for her contribution to manuscript preparation and proofreading; A. Benoit for technical assistance; N. Baumann and Q. LoGiudice for assistance with bioinformatics analyses; S. Roig Puiggros for help with schematic designs; all members of the Jabaudon laboratory and E. Azim, V. Castellani, A. Chédotal, C. Desplan and G. Pouchelon for constructive comments on the manuscript. The Jabaudon laboratory is supported by the Swiss National Science Foundation, the Carigest Foundation, the Société Académique de Genève FOREMANE Fund, and the Simons Foundation for Autism Research. E.K. is supported by a grant from the Machaon Foundation. U.T. is supported by Swiss National Foundation Synapsy (grant 51NF40-185897). J.P. is supported by the Public Instruction Department, Geneva. J.M.K. was supported by Boehringer Ingelheim and Jane Coffin Childs postdoctoral funds. D.H. and G.L.G. are supported by the International Foundation for Paraplegia Research and the Swiss National Foundation. A.S. and R.J.P. are supported by the Swiss National Science Foundation (SNF 31003A_175830), ETH Zurich Research Grant (ETH-27 18-2), EMBO Young Investigator Program (4217), Personalized Health and Related Technologies (PHRT), and National Centres of Competence – Molecular Systems Engineering. A.D. was supported by the Swiss National Foundation Synapsy (grant 51NF40-158776). C.B. is supported by the Swiss National Foundation (grant: 31003A_182326) and NCCR-Synapsy.

Author information

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Authors

Contributions

U.T., A.C., G.L.G. and S.F. performed the experiments. E.K. and J.P. performed the bioinformatics analyses. J.M.K. provided Sindbis virus and shared expertise on MAPseq. A.S. and R.P. designed, cloned and validated sgRNAs and Cas9 vector. E.K. and D.J. wrote the manuscript. U.T., A.C., G.L.G., S.F., J.M.K., D.H., C.B. and A.D. revised and edited the manuscript. We dedicate this manuscript to the memory of Alexandre Dayer, who passed away before completion of this work.

Corresponding author

Correspondence to Denis Jabaudon.

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The authors declare no competing interests.

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Peer review information Nature thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 MAPseq experimental procedure to study postnatal emergence of inter-areal cortical connectivity.

a, Left, retrograde labeling from M or S2 using green (Gbeads) or red (Rbeads) retrobeads. *Injection site. Center, Gbeads M- and Rbeads S2- inter-areal cortical projection neurons (\(\vec{{\rm{M}}}\) and \(\vec{{\rm{S}}2}\) ICPN) in S1 at P7 (arrowheads, retrogradely-labeled cells). Right,  Illustrations of \(\vec{{\rm{M}}}\) and \(\vec{{\rm{S}}2}\) ICPN layer positions at P5, P7 and P14 (P5: n = 4, P7: n = 3, P14: n =2 pups from 2 litters / target; 50 random quantified cells were plotted per condition; dot shades represent the different pups). b, Double retrograde labeling using Gbeads in M and alexa 546-conjugated CTB in S2 at P5, P7 and P14 (P5,P14: n = 4; P7: n = 3 pups from 2 litters; n = 50 to 200 \(\vec{{\rm{M}}}\) and \(\vec{{\rm{S}}2}\) ICPN per pup). Note that the proportion of co-labeled cells (i.e. cells projecting to both M and S2) is stable from P5 on (two-way ANOVA test: P value > 0.9999). c, Left, MAPseq principle. Center, infected neurons in S1 expressing Sindbis-GFP 14 hours after infection. Arrowhead shows an axon labeled with GFP. Right, in situ hybridization at P14 shows barcode-Gfp mRNA (level 2 from Fig. 1b). d, Microdissections of injection and target sites. e, S1 ICPN multiplex projections at P5, P7 and P14 (showing data from Fig. 1c as percent max projection). f, Diversity of projection patterns measured by the entropy at each age (see Methods). In black is the value for random patterns (i.e. 6 bits / pattern). g, Similarity between projection pattern matrices, shown as 1 - relative Kullback-Liebler divergence value (see Methods). Note that P7 and P14 projection pattern matrices display the highest similarity. h, First and second targets of ICPN at P5, P7 and P14. ICPN with only one target are represented with same first and second targets. Values are shown as mean ± s.e.m. (b, h). Scale bars, 100 μm (a, b); 300 μm (c); 1 mm (d). A, auditory cortex; C, contralateral cortex; CTB, cholera toxin B; Gbeads, green retrobeads; Hip, hippocampus; M, motor cortex; P, postnatal day; Rbeads, red retrobeads; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; Sub, subcortical; Str, striatum; Thal, thalamus; V, visual cortex.

Extended Data Fig. 2 \(\overrightarrow{{\bf{M}}}\) and \(\overrightarrow{{\bf{S}}{\bf{2}}}\) ICPN have otherwise similar multiplex projection patterns.

a, Cluster analysis of \(\vec{{\rm{M}}}\) and \(\vec{{\rm{S}}2}\) ICPN projecting in more than one target at P14 reveals 4 projection patterns (n = 400 \(\vec{{\rm{M}}}\,\)or \(\vec{{\rm{S}}2}\) ICPN). Note the similar distribution of patterns for both populations. b, Distribution of P5 and P7 \(\vec{{\rm{M}}}\) and \(\vec{{\rm{S}}2}\) ICPN projecting in more than one target in the P14 clusters using k-nearest neighbors (knn; see Methods). Note that \(\vec{{\rm{M}}}\) and \(\vec{{\rm{S}}2}\) ICPN show similar patterns at P7 and P14.

Extended Data Fig. 3 ConnectID data preprocessing and quality controls.

Data presented here are the raw data from the single-cell analysis, and include cells without detected projections in the targets. a, Data processing allowing retrieval of single-cell gene expression and projection(s). b-f, Non-filtered barcodes (BC) quality controls in single cells after sequencing error correction (step 5 in a) for each pup used in this study (n = 1859 cells). b, Single cells and their corresponding BC. Each column corresponds to a cell, and each line to a BC sequence. c, BC sequence(s) within cells. Number of BC with distinct sequences per cell. Pie chart represents summary of the data. ~80% of the cells express 1 to 4 BC sequences. d, BC counts for the top 5 most expressed BC. Box plots indicate median ± s.d. and interquartile range. e, Counts of the most expressed to the less expressed BC for cells with more than 1 BC sequence. f, Barcodes across cells. Number of BC sequences found in the top 9 cells (left). 80% of the BC sequences are found in only one cell. Counts for the BC found in several cells from the cell where it is the most abundant to the cell where it is the least abundant (right).

Extended Data Fig. 4 Quality controls of single cells collected 14 hours after Sindbis infection.

a, Fluorescence activated (FAC) sorting of Sindbis-GFP+ / Hoechst+ cells (left), capture and quality controls of single cells in microfluidic wells using brightfield imaging (right). Wells with no cell, several cells, debris or with high mitochondrial reads (> 30% total mapped reads) were excluded. b, Number of mapped reads, of expressed (exprs.) genes, and proportion of mitochondrial (mito.) reads (on total mapped reads) per cell. Bottom right, note that the proportion of mitochondrial reads is not correlated with the viral load (i.e. with the number of Sindbis reads) in each cell. Retrogradely-labeled ICPN (Rbeads) collected at P9 were used as control cells for the effect of the viral infection. c, Viral load increases with the number of barcode(s) per cell (n = 2450 cells). Box plots indicate median ± s.d. and interquartile range. d, Ordinal regression model identifies genes with the strongest weight in distinguishing neurons with a higher viral load from those with a lower viral load. e, “least square” fit regression was performed to the expression set using viral load and number of expressed genes as variables to regress. rpm, reads per million.

Extended Data Fig. 5 S1 L2/3 ICPN have highly similar transcriptional identity.

a, Left, UMAP of SSp L2/3 intratelencephalic (IT) neurons and clusters from Yao et al., 202115. Right, distribution of cells across clusters. b, Distribution of neurons projecting to MOp (corresponding to “M”) or SSs (“S2”) retrogradely labeled (using retrograde AAV2 virus) in the UMAP (left) and within the clusters (right). Note that neurons projecting to MOp or SSs neither belonged to distinct transcriptional cluster nor clustered apart in the L2/3 IT Otof 5 cluster, suggesting that these subtypes have highly similar transcriptional identities. c, Top, tSNE representation of P14 neurons after layer microdissection. Bottom, main target and distribution of ConnectID neurons in superficial (SL) versus deep (DL) layers. d, Kmeans clustering of single cells based on their tSNE values (left) and distribution of cells in the 4 identified clusters (right). Note that cells cluster based on their age of collection. e, Kmeans clustering based on tSNE values calculated independently at each collection age (P7, P9, P14), and distribution of neurons with known projection within these clusters. P7 and P14 cells are from ConnectID experiments, while P9 cells are from both ConnectID and Rbeads experiments (see Methods). Note that developing ICPN are not detectably clustered by their axonal target, but rather by their layer position, as showed at P14 when SL and DL were microdissected. IT, intratelencephalic; L, layer; MOp; primary motor cortex; SSp, primary somatosensory cortex; SSs, secondary somatosensory cortex.

Extended Data Fig. 6 Transcriptional maturation of \(\overrightarrow{{\rm{S}}2}\) and \(\overrightarrow{{\rm{M}}}\,\) ICPN.

a, Pseudo-maturation score calculated for each cell (see Methods). b, Expression and number of genes in each wave for \(\vec{{\rm{S}}2}\) and \(\vec{{\rm{M}}}\,\) ICPN. c, Gene ontology analysis of genes belonging to each wave in \(\vec{{\rm{S}}2}\) and \(\vec{{\rm{M}}}\,\) ICPN. d, Venn diagrams showing shared genes between ontologies. e, Distribution of \(\vec{{\rm{S}}2}\,\) ICPN wave-defined genes into \(\vec{{\rm{M}}}\,\) ICPN waves (left), and of \(\vec{{\rm{M}}}\) ICPN wave-defined genes into \(\vec{{\rm{S}}2}\,\) ICPN waves (right) by gene ontology. f, Top, expression of axon development- (left), and dendrite- (center) related genes along pseudo-maturation in \(\vec{{\rm{S}}2}\,\) and \(\vec{{\rm{M}}}\) ICPN. Bottom, number of genes related to synapse in each wave for \(\vec{{\rm{S}}2}\) and \(\vec{{\rm{M}}}\) ICPN (left) and synapse-related gene expression along pseudo-maturation in \(\vec{{\rm{S}}2}\) and \(\vec{{\rm{M}}}\,\) ICPN (center) (repeated measures-ANOVA based on a general linear model with Geisser-Greenhouse correction). Right, examples of dynamics of gene expression for 3 classical axon (Chl1 and Tubb5) and dendrite (Map2) development-related genes. Dots correspond to single cells.

Extended Data Fig. 7 Functional maturation of \(\overrightarrow{{\rm{M}}}\) and \(\overrightarrow{{\rm{S}}2}\) inter-areal cortical projection neurons using calcium imaging upon somatosensory stimulations.

a, Calcium wide-field imaging upon sensory stimulations in Cux2:Cre x GCaMP6s anesthetized pups at P9 and P15. Experimental paradigm and in situ hybridization for the Cre transcript in Cux2:Cre P28 pup (from the Allen Brain Institute database). b, Pooled responses to whisker pad, hindlimb and forelimb stimulations in S1, M, S2, and V at P9 and P15. At P9, response in M was not different than response in V (considered as background value) (n = 3 animals / age; two-way ANOVA with Sidak’s multiple comparisons test: M versus V: P9: adjusted P value = 0.8421; P14: adjusted P value = 0.0001; S2 versus V: P9: adjusted P value = 0.0174; P14: adjusted P  value = 0.0123). Thinner traces represent the values for each individual pup. Grey, stimulation. Pooled responses are either represented by age (top) or by cortical area (bottom). Note the distinct response dynamics at P9 and P15, P15 responses decreasing faster after the stimulation than P9 responses. c, Response to whisker pad (top), hindlimb (middle), and forelimb (bottom) stimulations. The absence of response in M at P9 does not depend on the type of stimulation. Scale bar, 100 μm (a, top). Fluorescence (F) in targets was normalized to the mean fluorescence in S1. A, auditory cortex; BF, barrel field; FL, forelimb; HL, hindlimb; M, motor cortex; P, postnatal day; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; V, visual cortex.

Extended Data Fig. 8 Restricted up- and down-regulation of SOX11 in time and space alters intracortical connectivity.

a, Sox11 is transiently enriched in \(\vec{{\rm{S}}2}\,\) ICPN at P7 (Kolmogorov-Smirnov test: P value < 0.0001), as confirmed at the protein level in \(\vec{{\rm{S}}2}\) compared to \(\vec{{\rm{M}}}\) retrogradely-labeled ICPN (n = 3 pups from 2 litters per target; unpaired t-test: P value = 0.0149). Values are shown as mean ± s.e.m. b, Double-UP plasmid control (Ctl) / Sox11 constructs and Cre-dependent recombination allowing the expression of Scarlet (Ctl) or Scarlet + SOX11 (SOX11). c, Left, GFP and Scarlet expression at P14 without (no Cre) or after AAV-pCMV-Cre stereotaxic injection at P0 in putative S1 (pS1). Note the restricted expression of Scarlet in S1 cells, while GFP electroporated cells are found both in S1 and S2. Right, overexpression of SOX11 protein in Scarlet+ cells at P5. d, Quantification of the number of Cre+ cells in S1 at P17 (data are the mean values over 3 sections per pup; unpaired t-test: P value = 0.0808). Note that SOX11 overexpression postnatally does not affect neuron migration or survival. e, Left, representative images of Scarlet axons in C and CC. Center, fluorescence-based quantification of the area covered by axonal signal (normalized by the number of Cre+ cells in S1) in M, S2, C and CC for each pup (log10 values, see Methods; M: n = 13 Ctl, n = 13 SOX11; S2: n = 13 Ctl, n = 11 SOX11; C: n = 7 Ctl, n = 6 SOX11; CC: n = 11 Ctl, n = 11 SOX11 pups from 4 litters per condition). Number of brains analyzed differ across targets because in some cases no quantification was possible. Pink dashed line, threshold below which no axon was detected. Right, heatmap of axon signal intensity in M and S2 for each pup. Dark grey, pups for which no axon was detected in the target. Only brains in which S2 and M projections could be assessed are shown here (in two SOX11 cases, only M projections could be assessed). f, Co-culture of SOX11-overexpressing and control L2/3 neurons. Left, experimental procedure. Half of the embryos were electroporated with dUP-Sox11 alone (Ctl) and the other half with dUP-Sox11 and pCAG:Cre plasmids (SOX11) at E15.5. Control and SOX11 cortical neurons were dissociated and cultured together at E16.5. Right, at DIV2, axon length was similar in both conditions. Values are shown as mean ± s.e.m. (unpaired t-test: P value = 0.3101). g, Left, inducible single-guide (sg) RNA against Sox11 leads to absence of SOX11 expression after AAV-pCMV-Cre stereotaxic injection at P0 in putative S1. Right, representative images of Scarlet+ axons in each target and quantification of the axonal phenotype. Scale bars, 100 μm (c, e, g right); 10 μm (a, f, g left). d, e, g, Box plots indicate median ± s.d. and interquartile range. e, g, Two-way ANOVA with Sidak’s multiple comparisons test: Ctl versus SOX11: M: adjusted P value < 0.0001; S2: adjusted P value = 0.0008; M versus 2: Ctl: adjusted P value = 0.3186; SOX11: adjusted P value = 0.0251; Ctl versus Sox11 sgRNA: M: adjusted P value = 0.0833; S2: adjusted P value = 0.0339. CC, corpus callosum; DIV, days in vitro; dUP, double-UP; E, embryonic day; epor., in utero electroporation; M, motor cortex; L, layer; P, postnatal day; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; sfGFP, super-folded GFP.

Extended Data Fig. 9 Altered intracortical somatosensory connectivity impairs exploratory behavior.

a, Trajectories in open-field arena foreach pup. The name of each pup is indicated (#). Two different arenas were used accounting for slight differences in trajectory shapes. b, SOX11-overexpressing (SOX11) pups display decreased velocity (unpaired t-test: P value = 0.0355), decreased rearing (unpaired t-test: P  value = 0.0007), increased time immobile (Mann Whitney test: P  value = 0.0005) but similar time spent in the center of the open-field (unpaired t-test: P value = 0.9030) or time spent grooming (unpaired t-test: P value = 0.1990), compared to control pups (Ctl) (n = 12 Ctl pups; n = 13 SOX11 pups). Box plots indicate median ± s.d. and interquartile range. c, Feature plots of specific behavioral and anatomical parameters. d, Correlation between principal component (PC) 1 and the Scarlet axonal signal measured in M for each pup.

Supplementary information

Supplementary Information

This file contains Supplementary Notes 1, 2, including two Supplementary Figures.

Reporting Summary

Supplementary Table 1

ICPN used for MAPseq analyses. n = 140 (P5) or 400 (P7; P14) ICPN barcodes (see Methods) per pup (n = 4 pups for each age) were randomly selected to avoid any bias due to variability in Sindbis injection site depth. Barcode names are as follows: name of the pup, error-corrected sequence of the barcode, barcode counts and cluster information in S1 injection site (barcode counts before sequencing error correction, number of barcode sequences with less than 3 mismatches, error-corrected barcode counts; see Methods for normalization of barcode counts in targets).

Supplementary Table 2

Distribution of genes in the three transcriptional waves for \(\vec{{\rm{S}}2}\) and \(\vec{{\rm{M}}}\) ICPN. Normalized transcriptional patterns were clustered in 3 groups (waves) based on their distances along pseudo-maturation using pam function of cluster package (k = 3). The average expression pattern was calculated for each cluster and the distances of all transcriptional patterns to this average were calculated. Only genes closely related to a given pattern in both \(\vec{{\rm{M}}}\) and \(\vec{{\rm{S}}2}\) ICPN were kept for further analyses (n = 1,293 genes). Genes in each wave and their ontologies are indicated for both \(\vec{{\rm{S}}2}\) and \(\vec{{\rm{M}}}\) ICPN.

Supplementary Table 3

Neuron differentiation-, dendrite-, axon development- and synapse- related genes. Gene sets were established using QuickGO gene ontology annotations from EMBL-EBI.

Supplementary Table 4

Differential gene-expression analysis between \(\vec{{\rm{S}}2}\) and \(\vec{{\rm{M}}}\) ICPN at P7. Distances between \(\vec{{\rm{M}}}\) and \(\vec{{\rm{S}}2}\) ICPN gene-expression profiles along pseudo-maturation were calculated for all genes from wave 1 (that is, genes dynamically regulated along postnatal development, with high expression at P7). Genes with distances > 5.8 between \(\vec{{\rm{M}}}\) and \(\vec{{\rm{S}}2}\) ICPN were selected for ontology analyses using GSEA.

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Klingler, E., Tomasello, U., Prados, J. et al. Temporal controls over inter-areal cortical projection neuron fate diversity. Nature 599, 453–457 (2021). https://doi.org/10.1038/s41586-021-04048-3

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