A Translaminar Genetic Logic for the Circuit Identity of Intracortically-Projecting Neurons

bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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.

Title A translaminar genetic logic for the circuit identity of intracortically-projecting neurons

Authors

Esther Klingler1, Andres De la Rossa1,3, Sabine Fièvre1, Denis Jabaudon1,2

1Department of Basic Neurosciences, University of Geneva, Geneva, Switzerland.

2Clinic of Neurology, Geneva University Hospital, Geneva, Switzerland.

3Present address: Department of Cell Biology, University of Geneva, Geneva, Switzerland.

Correspondence should be addressed to D.J. ([email protected]).

Abstract

Distinct subtypes of intracortically-projecting neurons (ICPN) are present in all layers, allowing propagation of information within and across cortical columns. How the molecular identities of ICPN relate to their defining anatomical and functional properties is unknown. Here we show that the transcriptional identities of ICPN primarily reflect their input-output connectivities rather than their birth dates or laminar positions. Thus, conserved circuit-related transcriptional programs are at play across cortical layers, which may preserve canonical circuit features across development and evolution. bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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.

Introduction

Neurons of the neocortex are organized into six radial layers, which have appeared at different times during evolution, with the superficial layers representing a more recent acquisition. Input to the neocortex predominantly reaches superficial layers (SL, i.e. layers (L) 2-4), while output is generated in deep layers (DL, i.e. L5-6)1,2. Intracortical connections, which bridge input and output pathways, are key components of cortical circuits because they allow the propagation and processing of information within the neocortex, thereby transforming afferent signals into cortical output.

Two main types of intracortically-projecting neurons (ICPN) can be distinguished by their axonal features (Fig. 1a): (1) excitatory interneurons with short axons projecting locally within cortical columns, which are located in L4 and called spiny stellate neurons (SSN)3-6, and (2) excitatory neurons with long axonal projections, including callosally projecting neurons (CPN), which are found in both SL 6-8 and DL (CPNSL and CPNDL) . In addition to their distinct axonal features, neurons in these two classes can be distinguished by their hierarchical position within cortical circuits: SSN are the main recipients of thalamic input and project to both CPN and other SSN, while CPN connect with one another, but not back to SSN (Fig. 1a)4,5,9.

In this study, we investigate the molecular hallmarks that distinguish SSN,

CPNSL and CPNDL and relate their transcriptional signatures with their input-output connectivity (i.e. their "circuit identity"). Specifically, taking advantage of the presence of CPN in both SL and DL, we sought to identify lamina-independent genetic hallmarks of a constant circuit motif (i.e. interhemispheric connectivity) across distinct layers. Using retrograde tracing from the primary somatosensory cortex to label

contralateral CPN, we report that CPNSL and CPNDL are born at different times of corticogenesis and have distinct developmental histories. By performing three-way

unbiased transcriptomic comparisons between CPNSL, CPNDL and SSN, we find that circuit identity supersedes laminar identity in defining ICPN transcriptional diversity. Supporting the functional relevance of a primarily circuit-based transcriptional organization, overexpression of the SSN-specific transcription factor RORB was sufficient to reprogram the circuit identity of CPN within their original layer.

Together, these findings reveal a circuit-based organization of transcriptional programs across cortical layers, which we propose reflects an evolutionary bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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.

conserved strategy to protect canonical circuit structure (and hence function) across a diverse range of neuroanatomies.

Results

ICPN are developmentally and molecularly heterogeneous

In order to characterize the laminar diversity of ICPN, we labeled CPN in the primary somatosensory cortex by injection of fluorescent retrobeads in the contralateral hemisphere (Fig. 1b). Retrogradely-labeled cells had a bimodal spatial distribution in SL and DL, and were largely absent from L4, where SSN are located (Fig. 1b). This mutually exclusive distribution of SSN and CPN suggests shared lineage relationships between ICPN in which single subtypes are generated at a given time point of corticogenesis.

We next compared the developmental histories of CPNSL and CPNDL. Given their

distinct laminar location, CPNDL could either be born together with CPNSL and arrest

their migration within deep layers, or be born before CPNSL, together with other deep- layer neurons. To distinguish between these two possibilities, we determined the birthdates of the distinct ICPN subtypes by performing daily BrdU pulse-injections between embryonic days (E) 12.5 and E16.5, and retrogradely labeled CPN as

described above. This approach revealed that CPNDL are born at E12.5 and E13.5

(as are other DL neurons), while CPNSL are mostly born between E15.5 and E16.5

(Fig. 1c). Thus, despite similar contralateral projections, CPNSL and CPNDL have non-overlapping developmental (and potentially evolutionary) histories.

We next examined how select markers of distinct types of cortical neurons were expressed across these cells. For example, while SATB2 and CUX1 are

strongly expressed by CPNSL, but whether CPNDL and SSN also express these genes has not been systematically examined8,10,11. Using SATB2, CUX1, RORB (a L4 marker) and CTIP2 (a L5B corticofugal neuron maker) as canonical genes, we report overlap and heterogeneity in gene expression across ICPN (Fig. 1d). Most strikingly,

while all ICPN expressed SATB2, CPNDL neither expressed CUX1 nor CTIP2 (Fig. 1d). Thus, based on this select set of markers, and confirming and extending 8,10 previous results , CPNDL, CPNSL, and SSN constitute molecularly diverse and bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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.

partially overlapping populations of cells, which may be linked to their circuit properties.

Circuit identity supersedes laminar identity in defining ICPN transcriptional diversity

Molecular distinctions between the different types of ICPN could either reflect their

laminar identity or their circuit identity (Fig. 2a). In the layer-based scenario, CPNSL

and CPNDL are only remotely related, reflecting their distinct laminar locations and developmental origins, while in the second scenario, the shared circuit properties of

CPNSL and CPNDL are reflected in closely-related transcriptional programs, as has recently been reported in cortical interneurons12.

To distinguish between these two possibilities, we examined the genetic

signatures of CPNSL, CPNDL, and SSN using RNA sequencing (Fig. 2a). First, we

isolated CPNSL and CPNDL by using retrograde-labeling, laminar microdissection, and fluorescence activated cell sorting (FACS) at P10, a time at which interhemispheric 13 connectivity is largely achieved . Second, we isolated CPNSL from SSN by performing retrograde labeling and FACS in transgenic mice in which SSN specifically express tdTomato (Scnn1aTg3CrexAi14tdT mice5). Two-way

transcriptional analyses comparing CPNSL and CPNDL identified 136 differentially-

expressed genes, while comparison of CPNSL and SSN identified 204 differentially- expressed genes (Fig. 2b left, and Supplementary Fig. 1 and 2 and Supplementary Table 1), whose specificities were confirmed with in situ hybridization data (Fig. 2b right, and Supplementary Fig. 3). Identified genes included previously known markers such as Mdga1 and Pcp4, which were enriched

in CPNSL and CPNDL respectively, and Rorb, which was enriched in SSN (Supplementary Fig. 3). Interestingly, corticofugal neuron markers such as Fezf2

and Bcl11b were weakly expressed in CPNDL, consistent with the presence of striatal projections in at least a subset of these neurons14,15. Supporting the functional relevance of these transcripts, comparison of gene ontologies identified greater enrichment in transcripts related to neuron projections and activity/physiology-related

functions when comparing CPNSL with SSN, consistent with the distinctive circuit position and function of SSN within cortical circuits (Fig. 2c). bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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.

To assess the transcriptional relationship between CPNSL, CPNDL, and SSL, we performed unsupervised clustering of samples based on transcriptional

signatures. Hierarchical clustering revealed that CPNSL and CPNDL are more closely related to one another than to SSN (Fig. 2d). This suggests a primarily circuit-based organization of transcriptional programs. To formally demonstrate this possibility, we compared the discriminative power of the layer-based taxonomy to a circuit-based taxonomy, as previously described16. This quantitative assessment of these two taxonomies revealed that the circuit-based classification was more discriminative than the layer-based classification at all levels of stringencies examined (Fig. 2e, top). Accordingly, cell-type specific genes were more differentially expressed

between CPNSL and SSN than between CPNSL and CPNDL (Fig. 2e, bottom). Together, these data indicate that ICPN molecular identities more closely correspond to their circuit properties than laminar location.

RORB-overexpressing CPNSL acquire SSN-like circuit properties

Finally, we sought to identify a functional molecular counterpart to the circuit-based classification identified above, using Rorb as a proof-of-principle transcript. Indeed,

this orphan receptor shows a 3-fold enrichment in SSN vs. CPNSL (Supplementary Table 1) and has been implicated in circuit assembly within and beyond the cortex17- 19. Here, we directly examined the function of RORB in intracortical circuit assembly by assessing whether targeted overexpression in ICPN induces acquisition of SSN- type morphology, electrophysiology, and circuit connectivity. For this purpose, we

electroporated a plasmid coding for RORB at E16.5, the time of birth of CPNSL. As previously reported, a fraction of RORB-overexpressing cells showed migratory defects and did not reach the cortex17 (Supplementary Fig. 4), yet some cells

maintained their normal migration and reached superficial layers (ICPNRORB, Fig. 3a,

left and Supplementary Fig. 4). We first compared the morphology of ICPNRORB with

control cells born at E14.5 (SSN) or at E16.5 (ICPNL2/3). In contrast to ICPNL2/3, the SSN are characterized by the absence of an apical dendrite3,5,20. Strikingly, as it is

the case for SSN, ICPNRORB actively retracted their apical dendrite between P3 and

P7, which was not the case in control ICPNL2/3 (Fig. 3a, center and right and Supplementary Fig. 4). Thus, RORB expression controls acquisition of a key morphological feature of SSN. bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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.

We next examined whether ICPNRORB also acquired electrophysiological properties

of SSN, including changes in Ih-type cationic conductances and membrane excitability5,21. Consistent with acquisition of SSN-type electrophysiological features,

whole-cell patch-clamp recordings in acute cortical slices showed a lack of Ih currents

in ICPNRORB, as well as increased membrane excitability (Fig. 3b,c). Interestingly,

action potential duration was shorter in both SSN and ICPNRORB compared to

ICPNL2/3, which could account for the higher firing rate in the former cells (Fig. 3c). Thus, RORB expression controls acquisition of key electrophysiological features of SSN.

Finally, we examined whether ICPNRORB acquired an SSN-type circuit identity. Consistent with acquisition of SSN-type local axonal projections, long-range

projections were lacking in ICPNRORB, as previously reported (Supplementary Fig. 4)18. Focusing on local microcircuit properties, we next examined the local

connectivity of ICPNRORB. ICPNL2/3 normally receive strong input from other ICPNL2/3, 4,5,9 while SSNL4 do not receive ICPNL2/3 input . We therefore examined whether

ICPNRORB displayed SSN-type input properties, i.e. lacked ICPNL2/3 input (Fig. 3d).

To this end, we targeted channelrhodopsin 2 (ChR2) expression into deep ICPNL2/3 via in utero electroporation at E15.5 and recorded photo-induced post-synaptic - responses in superficial E16.5-born ICPNL2/3. In contrast to ChR2 ICPNL2/3 neurons, which all displayed synaptic responses following optogenetic stimulation of

homotypic neurons, only 6/14 ICPNRORB responded to ICPNL2/3 stimulation, with dramatically smaller amplitudes than in control cells (Fig. 3e). Together, these findings reveal acquisition of SSN-type morphological, electrophysiological and circuit

properties by ICPNRORB.

Our findings reveal a genetic organization of ICPN in which transcriptional programs more closely reflect circuit properties than laminar location or developmental origins. From a phylogenetic perspective, we find these results interesting considering that superficial cortical layers are a recent evolutionary acquisition of mammals1,2: this suggests either that a specialized progenitor class generates CPN throughout corticogenesis, or that a convergent evolution has occurred in deep and superficial layer neurons, in which similar molecular programs were selected for trans-callosal axon extension. Finally, using RORB as a proof-of- principle transcript, we show that ectopic expression of a single gene is sufficient to bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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.

orchestrate the coordinated acquisition of the morphological, physiological and circuit properties of another intracortically-projecting neuron subtype. Along with similar recent findings in inhibitory interneurons12, this suggests that circuit properties are critical end-point determinants of neuronal identity and the result of convergent molecular programs during neuronal differentiation.

Acknowledgements

We thank the Genomics Platform and FACS Facility of the University of Geneva, A. Benoit and T. Kastylevsky for technical assistance; and members of the Jabaudon laboratory as well as U. Tomasello, G. Limoni, I. Vitali and E. Azim for constructive comments on the manuscript. The Jabaudon Laboratory is supported by the Swiss National Science Foundation and the Brain and Behavior foundation; E.K. is supported by a grant from the Machaon Foundation.

Author contributions

E.K., A.D.l.R., and D.J. conceived the project and designed the experiments. E.K., A.D.l.R and S.F. performed the experiments. E.K. and D.J. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

References

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Materials and methods

Mouse strains.

C57Bl/6 male and female pups and adult mice were used. The Scnn1a-cre mouse line (Jackson Laboratories22; #009613) was crossed with CAG-tdTomato reporter mice (Jackson Laboratories; #007914). All experimental procedures were approved by the Geneva Cantonal Veterinary Authority and conducted according to the Swiss guidelines.

Plasmids.

We generated plasmids using a standard endotoxin-free Qiagen kit (#12362). The ChR2 T159C23 plasmid was subcloned into the pCAGIG_IRES_GFP vector. The pCBIG_Rorb_IRES_GFP plasmid was obtained from Addgene (#48709)17.

In utero electroporation.

Timed pregnant C57Bl6/J mice (Charles River Laboratory) were electroporated in utero at E14.5, E15.5 or E16.5 as previously described17.

BrdU pulse labeling.

A single dose of 50 mg/kg of animal weight of BrdU (16 mg/ml) was administered by an intraperitoneal injection in the mother during pregnancy, from embryonic day (E) 12.5 to E16.5.

Retrograde labeling.

Anesthetized pups were placed in a stereotaxic apparatus at postnatal day (P) 9

and injected with red Retrobeads™ IX from Lumafluor (for CPNSL vs. CPNDL comparison) or with Alexa-488 conjugated cholera toxin subunit B (CTB,

Invitrogen, #C-34775) (for CPNSL vs. SSN comparison) in S1 (200 nl; coordinates from the lambda: anteroposterior: 3 mm, mediolateral: 3mm).

Immunohistochemistry.

Postnatal mice were perfused with 4% paraformaldehyde (PFA) and brains were fixed overnight in 4% PFA at 4 °C. Eighty m vibrating microtome-cut coronal sections (Leica, VT1000S) were incubated 2h at room temperature in a blocking/permeabilizing solution containing 5% bovine serum albumin and 0.3% triton X-100 in PBS, and incubated for 2 days with primary antibodies at 4°C. bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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.

Sections were then rinsed three times in PBS and incubated with the corresponding Alexa-conjugated secondary antibodies (1:500; Invitrogen) for 2 h at room temperature. For immunohistochemistry against RORB, a pre-treatment in 10 mM sodium citrate, 0.05% tween 20 buffer (pH6) at 85°C for 40 minutes was performed before running the same protocol mentioned above of the procedure. For immunohistochemistry against BrdU, a pre-treatment with 2N HCl at 37°C for 40 minutes was performed. Primary antibodies and their dilutions were: mouse anti-human ROR (Perseus proteomics, # PP-H3925-00, 1:200), chicken anti-GFP (Invitrogen, # A10262, 1:2000), rat anti-BrdU (Abcam, # ab6326, 1:200), rat anti- CTIP2 (Abcam, # ab18465, 1:500), rabbit anti-CUX1 (Santa Cruz, # sc13024, 1:250), mouse anti-SATB2 (Abcam, # ab51502, 1:200).

Image acquisition and quantifications.

All images were acquired on a Nikon A1r spectral confocal microscope, equipped with 40x 0.6 CFI ELWD S Plan Fluor WD objective. Cells were selected for analysis only if there was no overlap with the primary dendrites of other labeled neurons. GFP/RORB overexpressing neurons were reconstructed for quantitative analysis of neuronal dendritic arborization using Imaris software. Quantifications of CUX1/SATB2/RORB/CTIP2 fluorescence intensity were done using Fiji software, by normalizing the row values to the background intensity (measured on a region of the section without positive cell) on each section and displayed them as the percent of max value on the section. For CTIP2 intensity quantification, the max value was measured in a positive L5 cell (since most ICPN express very low to not detectable levels of CTIP2).

To quantify BrdU+ CPN after injection of BrdU at different embryonic stages, we calculated the percentage of Retrobead-labeled CPN in L2/3, L5a, and L5-6 that displayed a high intensity of BrdU. Only sections with at least 10 Retrobead- labeled CPN per layer were kept for analyses (n=2 to 3 sections per pup, n=3 to 5 pups per age). For L4 BrdU+ cells, we delineated L4 with CUX1 staining, from which the barrels were identifiable, and quantified the percentage of BrdU+ cells in this area. For the heatmap in Fig 1c, the total number of BrdU+ ICPN was normalized to 100% per layer, showing the peak of birth for each ICPN population per layer. Error bars represent s.e.m.

Tissue microdissection, cell sorting and RNA sequencing. bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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.

Microdissection of primary somatosensory cortex (S1) from one litter (4 pups) corresponds to one biological replicate (n = 3 to 6 for each condition). Fresh coronal brain sections (600 μm) were cut on a vibrating microtome (Leica, VT1000S) and S1 was microdissected using a Leica Dissecting Microscope (Leica, M165FC) in ice-cold oxygenated artificial cerebrospinal fluid (ACSF) under RNase-

free conditions. For the comparison of SSN vs. CTB-labeled CPNSL, we used the same procedure as in ref. 10 for cell dissociation. For the comparison of

Retrobead-labeled CPNSL vs. CPNDL, cells were dissociated by incubating microdissected samples in 0.5 mg/mL pronase (Sigma, #P5147) at 37°C for 10 minutes, followed by incubation in 5% bovine serum albumin for 3 minutes and manual trituration in ACSF using pulled glass pipettes. Cells were then centrifuged for 10 minutes at 600 rpm and resuspended before filtration using a 70 m cell strainer (ClearLine, # 141379C). Cells were then incubated for 10 minutes at 37°C with Hoechst (0.1 mg/mL) and isolated using a Beckman Coulter Moflo Astrios FAC- sorter. Singlet Hoechst+ cells were sorted according to their Forward and Side scattering properties (FSC and SSC), and their negativity for Draq7TM (Viability dye, Far red DNA intercalating agent, Beckman Coulter, #B25595). RNA was extracted using an RNeasy kit (Qiagen, #74034). cDNA libraries were obtained using SMARTseq v4 kit (Clontech, # 634888) and sequenced using HiSeq 2500 sequencer.

Analyses.

For bulk RNA sequencing data analyses, we kept only genes expressed more than 20 rpm in at least one of sample. To perform three-way analyses on the layer vs.

circuit datasets, we normalized the expression of genes by the means of CPN SL samples from both experiments. For Fig.2d, samples with similar expression of genes and therefore similar principal components loadings, are most likely to localize near each other in the embedding24,25. Hierarchical clustering was performed using euclidian distance metrics. To compare the discriminative power of the layer-based classification (that is, SL vs. DL) with the circuit-based classification (that is, local vs. callosal), we used the same approach as described in ref. 16 (the paragraph below is directly modified from the original description in this study): we trained 2 linear nu-support vector machine (nu-SVM) classification models. Nu corresponds to the degrees of freedom of the SVM model, and thus bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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.

inversely correlates with stringency. We determined the maximal margin of separation between the two populations (that is, SL vs. DL CPN, or SSN vs.

CPNSL), which indicates how distinct these two populations are. Because the ‘nu’ parameter controls the stringency of the model, we confirmed the results using a range of nu values between 0.1 and 0.5. We looked for genes differentially expressed in the layer and the circuit models using SVM with nu=0.3. We considered as differentially expressed genes with a FDR < 0.1.

Statistical analyses of morphological and electrophysiological parameters were performed using Graphpad Prism software. For statistical analyses of neuron morphology at P7, we used 2-way ANOVA and multiple comparisons with

Bonferroni correction (n=17 ICPNL2/3; n=13 ICPNRORB; n=13 SSNL4). For analyses of electrophysiological parameters, we used Kruskal-Wallis non-parametric test with multiple comparisons when comparing the 3 ICPN populations and Mann-

Whitney test when comparing only ICPNL2/3 and ICPNRORB. The number of recorded cells is indicated on each figure. For statistical analyses of dendritic length at P21, we confirmed normality of the data using D’Agostino & Pearson normality test and performed unpaired t-test.

Electrophysiology.

300 µm thick coronal slices from P21 mice were cut in cooled ACSF containing

125 mM NaCl, 2.5 mM KCl, 1 mM MgCl, 2.5 mM CaCl2, 1.25 mM Na2HPO4, 26 mM

NaHCO3 and 11 mM glucose, oxygenated with 95% O2 and 5% CO2. Slices were kept at room temperature and allowed to recover for 1h before recording. Under low magnification, the barrels in L4 could be readily identified, and high-power magnification was used to guide the recording electrode onto visually identified neurons. Whole-cell voltage-clamp recordings were performed with 3–4 MΩ

electrodes filled with a solution containing 140 mM KCH3SO3, 2 mM MgCl2, 4 mM

NaCl, 5 mM P-creatine, 3 mM Na2ATP, 0.33 mM GTP, 0.2 mM EGTA and 10 mM HEPES adjust to 300 mOsm l-1 1 and pH 7.2 with KOH. Currents were amplified (Multiclamp 700B, Axon Instruments), filtered at 5 kHz and digitized at 20 kHz

(National Instruments Board PCI-MIO-16E4, Igor, WaveMetrics). Ih was measured in voltage clamp using a -40mV step (500 ms) and was calculated by the difference of current between the beginning and the end of the voltage step. The firing pattern was studied by current clamp recording of the neurons during the bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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.

injection of 20 to 300 pA of current (50 pA stepped) for 500 ms. Optogenetic stimulations were performed using 0.5 ms blue LED light pulses at 0.05 Hz in L2/3 ChR2-expressing sections, and photo-induced EPSCs were recorded from

ICPNL2/3 and ICPNRORB in presence of 10 µM bicuculline. No series resistance compensation was used. Values are presented as mean ± s.e.m.

22. Madisen, L. et al. Nat Neurosci 13, 133–140 (2009). 23. Berndt, A. et al. Proceedings of the National Academy of Sciences 108, 7595– 7600 (2011). 24. Macosko, E.Z. et al. Cell 161, 1202–1214 (2015). 25. Telley, L. et al. Science 351, 1443–1446 (2016).

Figure legends

Figure 1 Diversity of intracortically-projecting neurons (ICPN) across cortical laminae. a, Schematic representation of the 3 populations of ICPN: callosally-

projecting neurons from superficial (CPNSL) and deep layers (CPNDL) and spiny stellate neurons from layer (L) 4 (SSN). b, Distribution of CPN within cortical laminae at P11. CPN were labeled by injection of red Retrobeads (Rbeads) in the contralateral somatosensory cortex. CPN were absent from L4, where SSN are located. c, Birthdates of ICPN reflect the inside-out generation of cortical neurons. d, Expression of cortical markers by ICPN. Scale bars represent 150 m (b, c, d, left), 20 m (d, middle). DL: deep layers, SL: superficial layers.

Figure 2 Circuit properties are the primary determinant of transcriptional identity for

CPNSL, SSN, and CPNDL. a, Schematic representation of the experimental strategy

and working hypotheses. b, Three-way transcriptomic comparison between CPNSL,

SSN, and CPNDL reveals type-specific and shared genes (left). Examples of selected gene in situ hybridizations (ISH, source: Allen Brain Atlas) and pseudo-intensities of stacked ISH of all significant genes that were referenced in the database (right). c, Ontologies of differentially-expressed genes. d, Unbiased clustering delineates

CPNSL, SSN, and CPNDL. Circles represent individual samples. tSNE, t-distributed

stochastic neighbour embedding. e, SSN vs. CPNSL (circuit-based hypothesis)

classification is superior to CPNSL vs. CPNDL classification (layer-based hypothesis) at all levels of stringency (top). Circuit-specific genes are more differentially expressed than layer-specific genes (bottom). Genes with fold changes > 2 are highlighted in red. ***P<0.001; Welch’s two-sample t-test. Scale bar in b represents 150 m.

Figure 3 ICPNRORB acquire SSN-like morphology and electrophysiological/circuit

properties. a, In contrast to ICPNL2/3, RORB-overexpressing ICPNL2/3 (ICPNRORB, filled arrowheads) and SSN do not have an apical dendrite at P7. b, In contrast to

ICPNL2/3, ICPNRORB and SSN lack Ih currents. Sample traces show Ih current in

response to a 500 ms, -40 mV square voltage step. c, ICPNRORB acquire SSN-like excitability. d, Schematic representation of L2/3-L4 canonical microcircuit. e, bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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.

ICPNRORB display SSN-like cortical circuit features. Left, experimental design and illustration. Right, sample traces, percentage of response and response amplitude

following 0.5 ms light stimulation of ICPNL2/3 (blue arrowhead). Scale bars represent 50 m (a, top, f), 30 m (a, bottom), 25 m (b).

a b DAPI Rbeads Overlay Injection (n=7) CPN P9 P11 * L2/3 L2/3 SL Analysis 50.1% Contralateral hemisphere L4 L4 42.5% 7.4% L2/3 CPNSL CPN Superficial L2/3 * L4 L5 L5 DL layers (SL) L5a L2/3 L4 SSN L5a

Radial position L5b-6 L5 L6 L6 Deep Rbeads DAPI layers (DL) CPN L6 DL * injection site

0 0.01 0.02 Density c BrdU pulse at: CPN CPN L4 CPN CUX1 RbeadsBrdU E12.5 E13.5 E14.5 E15.5 E16.5 L5b-6 L5a L2/3 P11 BrdU CPN E16.5 L2/3 L2/3 SL E15.5 L4 L4 E14.5 CPN L5a L5 E13.5

BrdU pulse date CPN DL L5b-6 E12.5 L6 0 20 0 20 0 20 0 20 E12.5E13.5E14.5E15.5E16.5 BrdU+ cells (% total labeled cells) 0 100%

d SATB2 CUX1 RORB CTIP2 CUX1 SATB2 CTIP2 SATB2 CUX1 P11 CPN L2/3 L4 L2/3 CPN L5a SL SL CPN L5b-6 L4 0 50 100 0 50 100 L5a % max intensity

L5b RORB CTIP2 DL CPN L2/3 DL L4 L6 CPN L5a CPN L5b-6 Rbeads CUX1/SATB2/CTIP2 Hoechst 0 50 100 0 50 100

Figure 1 Diversity of intracortically projecting neurons (ICPN) across cortical laminae. bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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. a b Rbead labeled- Tomato L4 Ptprk Tnc Lypd1 Enc1 P14 Igfbp4 Nov Tnc Cckbr Kcnab3 Zmat4 Cox6a2 Kcnk2 Sema5a CPN SSN Efnb2 Nov Igfbp5 Efnb3 L2/3 Cux2 Mdga1 Wfs1 Dpysl5 L4 Gpr88 CPNSL Pcdh19 Chrm3 L5 Hypotheses: Igfbp4 Chrm1 Gria3 Chl1 12 L6 Layer-based Calb1 Gap43 Cdh13 identities Kitl CPN Fxyd6 Microdissection SL Stacks of ISH: CPN 136 CPNSL SSN CPNDL and FACS at P10 SL 54 SSN n=6 n=16 n=18 L2/3 SSN 47 CPNDL CPNDL 67 Bulk RNA Rorb Etv1 L4 sequencings Circuit-based Zmat4 Kcnk2 identities 3 L5 Ppp1r1b Fezf2 CPNSL Whrn CPNSL CPNSL CPN Kcnab3 Pcp4 DL Grm2 L6 vs CPNDL vs SSN Fos Rprm SSN 0 1 Cckbr Sema5a Intensity

40 c d e Circuit-based CPNSL CPNSL 30 vs. CPNDL vs. SSN CPNSL e Neuron projection CPN CPNSL 3 20 100 DL Response to external stimulus CPNSL d Layer-based CPN CPNSL 4 10 Cell growth 50 SL CPNSL 2 % of max. margin 0 Cell differentiation CPNSL 1 0.1 0.2 0.3 0.4 CPN 3 Cell adhesion 0 DL Nu (= 1 / stringency) CPNDL 2 Cell−cell signaling tSNE2 CPN 4 −50 DL 1.00 *** CPN 1 Synaptic transmission DL SSN a 0.75 Voltage−gated ion channel activity −100 SSN SSN b SSN c Glutamate receptor activity 0.50 −50 −25 0 25 1500 1000 500 0 GABA receptor activity (fdr < 0.1) tSNE1 Distance 0.25 5 0 5 10 15

-log2(FDR) Abs log2(fold change) 0.00 Layer- Circuit- based based

Figure 2 Circuit properties are the primary determinant of transcriptional identity for CPNSL, SSN, and

CPNDL. bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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.

a Apical ICPNL2/3 ICPNRORB P7 μ m) ROR P7 Merge 80 ICPNL2/3 40 L2/3 0

n = 16 Basal Dendritic length ( ** L4

ICPNRORB GFP ROR Hoechst GFP ROR Merge L2/3

n = 13 ICPN SSN RORB ICPN

n = 13 b c ICPNL2/3 ICPNRORB SSN

ICPNL2/3 ICPNRORB SSN Half-width

150 Amplitude 20 mV 5 ms 40 mV ** (pA) ude 100 ** 10 I 200 ms 3 * h ** mp li t 2 50 5 40 pA numbe r 1 200 ms AP absolute a h 13 14 9 Half-width (ms) I 0 0 0 12 129 L2/3 0 100 200 300 L2/3 RORB RORB SSN SSN ICPN Current Injection (pA) ICPN ICPN ICPN d e Input from ICPN 1000 L2/3 P21 Epor Tom Epor Rorb ICPN ICPN Recording of L2/3 L2/3 RORB ICPN ICPN + P21 L2/3 RORB Tom neurons *** 400 (pA) ude

? L2/3 SSN 50 pA mp li t

a 200 E16.5 200 ms

Epor Tom Ne t 14 14 vs. Rorb-Tom Percentage of response: 0 L2/3 100% 42.9% RORB ICPNL2/3 ICPNRORB E15.5 ICPN ICPN Epor ChR2 ChR2-venus Tom Biocytin n = 14 n = 14

Figure 3 ICPNRORB acquire SSN-like morphology and electrophysiological/circuit properties. ed genesaresignificantly differentially thepopulations. expressedbetween Supplementary Fig. 1 b a

Normalized expression in CPNDL Normalized expression in SSN 1.0 1.5 2.0 1.0 1.5 2.0 2.5 3.0 bioRxiv preprint 1.0 1.0 n=70 CPN n=66 CPN CPN SL vs.DL CPN SL n =154CPN n =50SSNenrichedgenes; Avp Gjc2 Tmem88b Syt6 Bmp4 Cyp26b1 Myrf Plxdc1 Nr4a2 Plekhg6 Lemd1 SL Adamts4 not certifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. Gjc3 Elovl7 Enpp6 Plxnb3 Col5a1 doi: Rnf122 vs.SSN Plch2 Tspan15 Ltbr Hdac7 Calb2 Lypd6b SL DL Sv2c Lims2 Trp53i11 Slit3 https://doi.org/10.1101/290395 Col6a1 Igsf1 Cntnap5b Cd163 Tmem163 Gjd2 Hcn4 Bcl11b Il33 Fezf2 Ldb2 Cacna2d2 Tgfbi Ankrd33b Cd36 Ccl7 Wnt4 Filip1 enrichedgenes Ccbe1 Gm15440 enriched genes; Ctsk Slc13a5 Hmox1 Lamb1 Dcn Normalized expressioninCPN 1.5 Igsf9 Lrrc55 Spsb1 SL Tppp3 Gal3st1 Plxnc1 F730043M19Rik Vip Ugt8a Lct Nptx2 Pf4 Gm26917 Arhgef28 Kcnab3 Cntn4 Lyve1 Kcnab1 enrichedgenes Igf1 Sema5a Grik1 Cpne9 Oxtr Gabrg3 Sema4g Gpr6 Differentialin CPN expression gene Cpne5 Tshz3 Il10ra Tmcc3 Bcl6 Chrm3 Gm12371 Cckbr Htr7 Scube1 Lypd6b Snx8 Gpc6 Grm4 Dse Normalized expressioninCPN Cacng4 Cox6a2 Cacna1h Ppp1r1b Lypd6 Hrh3 Ubash3b Tpbg Sstr1 Sema3c Nr3c2 Prss12 Col6a1 Grin2c Ptpru Mtss1 Agt Parm1 Nell1 Zmat4 Ccdc80 Kcng1 Lypd1 Tmem108 Igsf21 Tmem150c Tnc Shisa9 Rcn1 Gabrd Pcdh8 Lrrtm4 Sox5 Syt17 Cgref1 Ust Cntn6 Tenm1 Ttc14 Ramp3 Vat1l Cacna2d3 Stum Cd24a Rgma Eps8 Whrn Fmo1 Col1a1 Pcdh19 Lct Gm26917 Ier5 Lyz2 Cdo1 Col1a2 Nebl Fam84a F13a1 Myo5b Plb1 Sh3bp4 Rprm Hdac9 Ptprt Bcas1 Nrn1 Kcnab1 Slc40a1 Rasgrp2 Wfs1 Gad1 Oxtr Atp6ap1l Synpr Lrrtm3 Dgkb Fam19a5 Fam149a A830036E02Rik Ehbp1l1 Ngb Tmem132d Kndc1 Galnt9 Mtss1l Reln Fam126a Etv1 Prmt2 Atp2b4 Efnb3 Tmem145 Lypd6 Hs3st2 Rfx3 Hs3st4 1810041L15Rik B3galt2 Prss12 Ctsc Sla Tbc1d16 Rasgrf1 Ptpro Ptpru Tspan17 Trpc3 Me1 Gatm Synm Gpc5 Ccnd2 Ypel1 Camk2d Pcp4 Cdh13 Ccdc80 2.0 Serpini1 Fos Grm2 Igsf21 Spon1 March4 Tnc Limk1 Pcdh8 Lrrtm4 Rac3 Neurod6 Rgs6 Edil3 Ntsr2 Pde1a Pitpnc1 Fkbp1b Gabra3 Pcdh11x Stab1 Chn2 Mrc1 Ppp1r16b Gnb4 Cryab Dab2 Chrdl1 B3gat1 Whrn Mdga1 Gabra5 Malat1 Scn3b Fxyd6 Nefm Kcnip3 Met Akap12 Btbd3 Gpr17 Gria3 Fam124a Sema3a Sez6 Gucy1a3 1.5 3110035E14Rik Rab3c Snhg11 Luzp2 Fstl1 Igfbp4 Lamp5 Efnb2 Cnr1 Kcnk2 Chl1 Epb41l2 Gng7 Csf1r Igfbp5 Pou3f1 Crip2 ; Lmo7 Enpp2 this versionpostedMarch28,2018. Rorb Nov Meg3 Cntnap2 Plxna1 Grm2 Chst1 Asic2 Kcnf1 Abat Arl15 Mlip Kitl Sowaha Prkcg Npnt Enc1 Gpr88 Chgb 2.5 Mdga1 Nfasc Lpl Lmo3 Cnih3 Nefm Nefl Lars2 Pak6 SL Serpine2 Rn18s−rs5 Ptprk Dpysl5 Foxp1 Igfbp4 Lamp5 Olfm1 Calb1 Tubb2b Cacng3 Prkca SL Nrsn1 Pantr1 Ncald Nov Tubb3 Gap43 Rragd Dpysl3 Lynx1 Cux2 Npnt Chrm1 Cacna2d1 SL SL Nefl Rap1gds1 Grin2b 3.0 vs. SSN(a),andinSL vs. Vsnl1 Plcxd2 The copyrightholderforthispreprint(whichwas 2.0 DL CPN(b). Annotat- bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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. 1 d 2 4 3 e 2 3 1 4 a SL SL SL SL SL SL DL DL DL DL b SSN aSSN bSSNCPN c CPN CPN CPN CPN CPN CPN CPN CPN CPN Satb2 Pcdh11x Fam124a Igfbp5 Myo5b 1.70 Synm CPN Gucy1a3 Syt17 SL Arhgef28 Snx8 Chrm3 Hdac9 SSN Fam149a Fam126a Tmem150c Tpbg 1.65 Bcl6 March4 Ypel1 CPN Kcnf1 DL Arl15 Fstl1 Tnc Gm12371 Gpr6 Rfx3 Enc1 Cdo1 1.60 Gabra3 Luzp2 Ccnd2 Fam19a5 Edil3 Serpini1 Gria3 Nebl Dgkb Cntn4 Cpne5 1.55 Sstr1 Scn3b Rgs6 Pak6 Cntn6 log2(norm. exprs+1) Rasgrf1 Ubash3b CPN genes Pde1a Crip2 Chst1 Abat 1.50 Epb41l2 Sparcl1 Serpine2 Gatm Tubb3 Ncald Rab3c Plxnc1 Wnt4 B3gat1 Plxna1 Me1 Ptprt Cux1 Shisa9 Lpl Htr7 Cntnap5b 1.90 Tenm1 Calb2 Fxyd6 Cgref1 Sox5 Eps8 Ehbp1l1 Gap43 Prmt2 1.85 Sez6 Prkcg Gng7 Csf1r Tmem108 Fkbp1b Sv2c Cdh13 1.80 Pitpnc1 Chl1 Gpc6 Ust Npnt Prss12 F730043M19Rik Rcn1 Pcdh8 Lrrtm4 1.75 Gjd2 Nov Filip1 Ptpru Oxtr Ngb Stab1 Mrc1 Ccbe1 Ctsk 1.70 Ramp3 Lrrtm3 Fam84a Vat1l Nr3c2 Cacna2d2 Lamb1 Camk2d Stum 1.65 Parm1 Enpp2 Slit3 Gabrg3 Atp2b4 Kcng1 Trpa1 Pcdh19 Rac3 CPN genes Synpr SL Gabra5 Ptpro Gpc5 Rorb Lct Sema4g Dpysl3 1.9 Lypd1 Wfs1 Cacna1h Efnb3 Mtss1 Hcn4 Dpysl5 Rgma Cpne4 Tubb2b Rasgrp2 Eif2s3y Ddx3y Igfbp4 Mdga1 Met 1.8 Cd24a Tppp3 Lypd6 Lypd6b Tbc1d16 Mtss1l Gnb4 Cacng4 Kndc1 Limk1 Trpc3 Sla Ldb2 Tmcc3 B3galt2 1.7 Tspan17 3110035E14Rik Cox6a2 Sh3bp4 Etv1 Igsf21 Nefm Kcnab1 Nefl Spon1 Spsb1 Lims2 Cryab Gpr17 Sema5a 1.6 Akap12 Ntsr2 Kcnk2 Nfasc Plb1 Chn2 1810041L15Rik Rprm Gal3st1 Hs3st4 CPN genes Bcl11b DL Grin2c Neurod6 Ctip2 Igsf1 Trp53i11 Galnt9 Il33 Tmem88b Elovl7 Gjc3 Plxnb3 Myrf Bmp4 Bcas1 Rnf122 1.6 Tspan15 Gm15440 Agt Hs3st2 Fezf2 Tmem163 Gjc2 Syt6 Nr4a2 Ugt8a Pcp4 Adamts4 Enpp6 Fmo1 Sema3a 1.4 Chrdl1 Vsnl1 Lmo7 Cacna2d1 Prkca Chrm1 Cux2

Atp6ap1l Calb1 Mlip Pantr1 Cntnap2 Grin2b Rragd Ptprk 1.2 Cnr1 Cacng3 Sowaha Lamp5 Sema3c Kitl Efnb2 Asic2 Pou3f1 SL genes Gad1 Reln Tgfbi Igf1 Dab2 Dse Vip Ctsc Ccl7 Slc40a1 Lyz2 Hmox1 Col1a2 Dcn Col1a1 F13a1 Pf4 Cd163 Lyve1 Cd36 Foxp1 Cnih3 Plcxd2 Lmo3 Rap1gds1 Lynx1 Il10ra Avp Snhg11 Ttc14 Malat1 Meg3 Rn18s−rs5 Lars2 A830036E02Rik Btbd3 Rorb Ppp1r16b Grik1 Igsf9 Tshz3 Grm2 Chgb Nrsn1 Hrh3 Scube1 Grm4 Olfm1 Tmem132d Gpr88 Ier5 Whrn Gm26917 SSN genes Ppp1r1b Fos Ltbr Cyp26b1 Nrn1 Zmat4 Plxdc1 Cacna2d3 Nell1 Cpne9 Col5a1 Plekhg6 Kcnip3 Gabrd Lrrc55 Ankrd33b Slc13a5 Lemd1 Tmem145 Hdac7 Nptx2 Kcnab3 Plch2 Cckbr Ccdc80 Col6a1

0 1 Normalized expression

Supplementary Fig. 2 ICPN differentially expressed genes. a, Expression heatmap and unbiased clustering of all differentially expressed genes in CPNSL vs. SSN and SL vs. DL CPN datasets. b, Expression of the markers used for primary characterization of the 3 ICPN populations (compare with values for corresponding protein expression in Fig. 1d). bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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.

CPNSL enriched genes CPNDL and SSN enriched genes Igfbp4 Mdga1 Nov Prss12 Ptpru Tnc Whrn Grm2

L2/3 L2/3

L4 L4

L5 L5

L6 L6

SSN enriched genes Cacna2d3 Cckbr Col5a1 Col6a1 Cyp26b1 Fos Gabrd Grik1 Grm4 Hrh3 Kcnab3 Nrn1 Ppp1r1b Rorb Scube1 Zmat4

L2/3

SL ICPN enriched genes Cacna2d1 Calb1 Chrm1 Cnr1 Col6a1 Cux2 Efnb2 Fstl1 Gad1 Gpr88 Grin2b Kitl Lpl Ptprk Pou3f1

L2/3

CPNDL enriched genes Agt B3galt2 Bcl11b Cox6a2 Etv1 Fezf2 Foxp1 Grin2c Kcnab1 Kcnk2 Ldb2 Limk1 Lmo3 Neurod6 Nfasc Pcp4 Rprm Sema5a

L2/3

CPN enriched genes Abat Bcl6 Cacna1h Calb2 Cd24a Cdh13 Chl1 Chrm3 Cntn6 Dpysl5 Efnb3 Enc1 Fxyd6 Gabra3 Gabra5 Gabrg3 Gap43 Gnb4

L2/3

Gria3 Igfbp5 Lypd1 Nr3c2 Pcdh19 Pde1a Plxna1 Plxnc4 Rac3 Rasfr1 Sema4g Slit3 Sox5 Sstr1 Tubb3 Wfs1 Wnt4

L2/3

Supplementary Fig. 3 Expression of differentially expressed genes by ICPN. Data from Allen Brain Atlas database (http://developingmouse.brain-map.org/). Scale bar represents 100 μm. bioRxiv preprint doi: https://doi.org/10.1101/290395; this version posted March 28, 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. a Epor Gfp E16.5 Epor Rorb-Gfp E16.5 b Epor Tom E16.5 P7 P3 ICPN ICPN RORB L2/3 L2/3

Epor Rorb-Tom E16.5 L5

GFP DAPI Tom DAPI c Tom Rorb 50 d Epor Gfp E16.5 P21 ICPNRORB P7 40 ICPNL2/3 30 20

n dendrites 10 CC 0 Tomato Biocytin 0 50 100 150 200 250 Radius Epor Rorb-Gfp E16.5

Imaris reconstructions m) 6000 ***

4000

2000

GFP DAPI 0 1412 L2/3 RORB Total dendritic length ( μ ICPN ICPN

Supplementary Fig. 4 ICPNRORB aquire SSN-like morphology and circuit properties. a, ICPNL2/3 labeled by GFP after in utero electroporation at E16.5. A fraction of RORB overexpressing cells are abnor-

mally positioned (arrows), though a significant proportion reaches L2/3 (ICPNRORB). b, At P3, ICPNRORB

display an apical dendrite (arrows) as do ICPNL2/3. c, In adults, ICPNRORB lack an apical dendrite and

have a reduced number of dendrites as well as a reduced dendritic length compared to ICPNL2/3. Imaris reconstructions were performed on biocytin filled electroporated cells, followed by Sholl analy-

ses. d, At P7, ICPNRORB do not extend an axon through the corpus callosum (CC, arrowhead) in con- μ μ μ trast to control ICPNL2/3. Scale bars represent 150 m (a, b, d, top), 40 m (c), 20 m (d, bottom).