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Hominini-specific regulation of CBLN2 increases prefrontal spinogenesis

Abstract

The similarities and differences between nervous systems of various species result from developmental constraints and specific adaptations1,2,3,4. Comparative analyses of the prefrontal cortex (PFC), a cerebral cortex region involved in higher-order cognition and complex social behaviours, have identified true and potential human-specific structural and molecular specializations4,5,6,7,8, such as an exaggerated PFC-enriched anterior–posterior dendritic spine density gradient5. These changes are probably mediated by divergence in spatiotemporal gene regulation9,10,11,12,13,14,15,16,17, which is particularly prominent in the midfetal human cortex15,18,19,20. Here we analysed human and macaque transcriptomic data15,20 and identified a transient PFC-enriched and laminar-specific upregulation of cerebellin 2 (CBLN2), a neurexin (NRXN) and glutamate receptor-δ GRID/GluD-associated synaptic organizer21,22,23,24,25,26,27, during midfetal development that coincided with the initiation of synaptogenesis. Moreover, we found that species differences in level of expression and laminar distribution of CBLN2 are, at least in part, due to Hominini-specific deletions containing SOX5-binding sites within a retinoic acid-responsive CBLN2 enhancer. In situ genetic humanization of the mouse Cbln2 enhancer drives increased and ectopic laminar Cbln2 expression and promotes PFC dendritic spine formation. These findings suggest a genetic and molecular basis for the anterior-posterior cortical gradient and disproportionate increase in the Hominini PFC of dendritic spines and a developmental mechanism that may link dysfunction of the NRXN–GRID–CBLN2 complex to the pathogenesis of neuropsychiatric disorders.

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Fig. 1: Phylogenetic, transcriptomic and regulatory characterization of developmental PFC upregulation of CBLN2.
Fig. 2: SOX5 represses CBLN2 through Hominini-specific regulatory deletions.
Fig. 3: In situ genetic humanization of the mouse Cbln2 enhancer drives upregulation and ectopic laminar Cbln2 expression in the neonatal frontal cortex.
Fig. 4: Increased density of dendritic spines in the mPFC of hCbln2 E2 mice.

Code availability

All software and code used in this study are publicly available.

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Acknowledgements

We thank S. Bai, T. Nottoli and X. Xing for technical help with generating transgenic mice and genetically humanized mice; A. Sousa for providing tissue; B. Lorente-Galdos and G. Santpere for helping with data analysis; A. Duque for using equipment from MacBrainResource (MH113257); V. Lefebvre for providing floxed Sox5 mice; and the members of the Sestan laboratory for comments. This work was supported by the National Institutes of Health (HG010898, MH106874, MH106934, MH110926 and MH116488) and the Simons Foundation Autism Research Institute (SFARI) (572080; to N.S.). Additional support was provided by the National Institutes of Health T32 fellowship (MH18268 to K.P.), the National Science Foundation Graduate Research Fellowship Program (to S.K.M.), the Kavli Foundation and the James S. McDonnell Foundation (to N.S.).

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Authors

Contributions

M.S., K.P. and N.S. designed the research. M.S. and K.P. performed the overall experiments and analysed the data. S.K.M. analysed the RNA sequencing, ChIP sequencing and genomic sequence data. M.S. generated the transgenic and knock-in mice. N.K. performed retrograde neuronal tracing with the AAV and analysed the data. Y.M.M. performed and analysed the electron microscopy experiments. X.C. and S.G.W. performed and analysed the preliminary electrophysiological analysis. N.S. conceived the study. M.S., K.P. and N.S. wrote the manuscript. All authors discussed the results and implications and commented on the manuscript at all stages.

Corresponding author

Correspondence to Nenad Sestan.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Spatiotemporal expression of CBLN2 in the human and macaque neocortex.

ac, Spatiotemporal expression of CBLN2 expression in the human and macaque cerebral cortex during development base on BrainSpan (brainspan.org) and PsychENCODE (development.psychencode.org, evolution.psychencode.org) human and macaque RNA-seq data15,20. The RNA-seq data consisted of tissue-level samples comprising the pial surface, marginal zone, cortical plate (layers 2-6) and adjacent subplate zone, of eleven prospective neocortical areas. Red and blue lines indicate human and macaque PFC CBLN2 expression, respectively, and dotted lines represent the non-PFC CBLN2 expression. Vertical grey box demarcates mid-fetal developmental periods. Predicted ages, timeline of human and macaque development and the associated periods are shown below. Visual representation of CBLN2 in human cortex with heatmaps of regional expression in human and macaque below during period 4-6 (b) and period 12-13 (c). Darker reds represent high expression levels. d, Anteroposterior visual representation of human CBLN2 expression at PCW 21 from the BrainSpan human prenatal laser microdissection microarray data (brainspan.org)37.

Extended Data Fig. 2 Expression profile of CBLN1,3,4 genes related to dendrite and synapse development and CBLN2 binding partners in macaque and human.

a, Developmental trajectory of CBLN2 expression compared to the expression of key genes related to dendrite development (i.e., MAP1A and CAMK2A) and synapse development genes (i.e., SYP, SYPL1, SYPL2 and SYN1). The lists of genes related to synapse and dendrite development were previously complied and analyzed for their expression trajectories by Kang et al.35. b, Developmental trajectory of CBLN2 expression compared to the expression of genes encoding CBLN2 binding partners (i.e., GRID1, GRID2, NRXN1, NRXN2 and NRXN3). Left and right panels show gene expression in human and macaque, respectively. Gene expression in PFC and non-PFC are indicated by solid and dashed line, respectively. Vertical grey box highlights the mid-fetal developmental periods. Predicted ages, timeline of human and macaque development and the associated periods are shown below. Gene expression values are represented as log2(RPKM+1). For all of these plots, the shading around the lines represents the 95% confidence interval. Abbreviations are as described in Fig. 1 legend.

Extended Data Fig. 3 Mouse Cbln2 expression at multiple stages and Cbln2 E2 transgenic lines.

a, b, Visualization of Cbln2 expression in wholemount (a) and sagittal sections (b) at PCD 11.5, 13.5, 15.5 18.5 and PD 4, 14 from Allen Brain Atlas developing mouse brain atlas (developingmouse.brain-map.org)71. Arrowheads highlight early rostral expression, UL, upper layer; L5, layer 5; SP, subplate. c, β-Galactosidase activity in two additional mouse brains from independent transgenic mouse carrying Cbln2 E2 conjugated with lacZ reporter at PCD 17. See Fig. 1c for the third replicate. FP, frontal pole; OB, olfactory bulb. Scale bars, 1 mm.

Extended Data Fig. 4 Comparative analysis of CBLN2 E2 deletions across mammals.

a, Schematic representation of CBLN2 E2 from mouse and primate species including apes (human, common chimpanzee, bonobo, gorilla, orangutan and gibbon), old world monkey (Rhesus macaque), new world monkey (marmoset) and prosimians (tarsier and lemur). HSD1, HSD2 and I3 are shaded. Putative RAR-RXR tandem binding sites indicated as red lines and putative SOX5-binding sites as blue lines. b, Sequence alignments of HSD1, HSD2 and I3 from macaque, mouse, and rat. Putative SOX5 binding sites are shown in red boxes. c, Sequence alignments of HSD1 and HSD2 from primates shown in a.

Extended Data Fig. 5 Conservation of CBLN2 E2 and deleted regions across species.

Phylogenetic tree of selected chordates including placental and non-placental mammals with information about presence of the CBLN2/Cbln2 gene and CBLN2 E2/Cbln2 E2. The last three columns describe the presence of HSD1, HSD2 and I3. The mouse E2 sequence was searched in each most updated animal genome browser at UCSC genome browser (as of December 22, 2019). E2 conservation criteria are: 1) identity over 80%; and 2) alignment length over 600 bp compared with mouse E2 (1005 bp).

Extended Data Fig. 6 SOX5 directly suppresses RXRG-RARB responsive human, chimpanzee, gorilla and mouse Cbln2 E2 enhancer.

a, Luciferase assay in Neuro-2a cell line from luciferase reporters conjugated to chimpanzee and gorilla wild CBLN2 E2. Two-tailed Student’s t-test; *P = 0.01; ***P = 9e-5 (Chimpanzee), 1e-5 (Gorilla); NS, not significant. Error bars; S.E.M.; N = 3 per condition. b, Overexpression of human SOX5 exerts a similar effect to mouse Sox5 on human and mouse Cbln2 E2 reporters. Two-tailed Student’s t-test; *P = 5e-3; ***P = 4e-5; ****P = 2e-6; 2e-6, 1e-6 (Human SOX5 + mouse Cbln2 E2); NS, not significant; Error bars, S.E.M.; N = 3 per condition. c, Cbln2 expression is upregulated in Sox5 conditional knockout brain at PD 0. Additional two replicates (Repl #2 and 3) not shown in Fig. 2d are shown. Scale bar, 200 μm (left); 100 μm (right). d, Constructs used for luciferase assay and generation of transgenic animals shown in e, f. e, Transgenic mouse brain at PCD 17 carrying Cbln2 E2 (N = 3) or Cbln2 E2 Fr1-lacZ reporters (N = 3). Cbln2 expression in the PFC is indicated by arrowheads. Endogenous Cbln2 expression is also shown for comparison (N = 4). Scale bar, 200 μm. f, Luciferase assay for Cbln2 E2 and Cbln2 E2-Fr1. Two-tailed Student’s t-test; **P = 1e-4 (Cbln2 E2), 2e-4 (Cbln2 E2-Fr1); ***P = 8e-5; NS, not significant; Error bars: S.E.M.; N = 3 per condition.

Extended Data Fig. 7 Humanized Cbln2 E2 knock-in mouse shows increased Cbln2 expression in neonatal neocortex.

a, Positions of single-guide RNAs (sgRNA1 and 2) to introduce double-strand breaks in the genomic DNA and targeting vector to replace WT mouse Cbln2 E2 region with that of human CBLN2 E2 (humanized hCbln2 E2) are shown. b, Genotyping strategy for F1 mice. Germline transmission in the F1 generation was confirmed by nested PCR using the primer set of mP3/mP4, followed by hP1/P2 as indicated in a. Two founders #13 and #25 were obtained. c, Mice in the following generation were genotyped by PCR with hP1/P2 and mP1/mP2. An example of genotyping for F2 of line #13 mice is shown (c). d, Comparison of Cbln2 expression between WT Cbln2 E2 (Mm;Mm) and hCbln2 E2 (Hs;Hs) neocortex at PD 0 using quantitative reverse transcription-PCR. RNA was extracted from the neocortex following the removal of hippocampus, olfactory bulb and subpallial regions. Two-tailed Student’s t-test; *P = 0.007; Error bars: S.E.M.; N = 5 per genotype. Genotyping were repeated at least two times in b and c.

Extended Data Fig. 8 Additional replicates.

Additional replicates from Fig. 3. a, b, The neocortex of the humanized hCbln2 E2 knock-in prenatal and neonatal mice exhibits upregulated Cbln2 in both upper and deeper layers compared to mice carrying WT Cbln2 E2. Additional two replicates (Repl #2 and 3) not shown in Fig. 3a are shown for PND 16 and PND18. Scale bars, 200 μm (left); 100 μm (right). All analysis and three replicates for PD1. Neocortex was divided into six equal bins spanning from pia to ventricular zone, and Cbln2 signal intensity was quantified for each bin and compared between WT and hCbln2 E2 (Hs;Hs). Two-tailed Student’s t-test; *P = 0.02 (PCD18), 0.02, 0.04 (PD1), **P = 1e-3 (PCD18), 1e-3, 3e-3, 3e-3 (PD1), ***P = 1e-4, 2e-4 (PCD18); N = 3 per genotype. Scale bars, 100 μm. c, Expression of the upper layer marker, Cux2, and SOX5 in adjacent tissue sections were detected by in situ hybridization and immunostaining, respectively. Scale bars, 100 μm. N = 3.

Extended Data Fig. 9 Expression of Sox5 in the developing mouse and human neocortex.

a, Double immunofluorescent staining for SOX5 and BCL11B in PD 0 and PD 7 mouse neocortex. N = 3 per condition. Scale bar, 100 μm. b, SOX5 expression in the PFC and non-PFC areas of the cerebral cortex of human and macaque across development. Red and blue lines indicate human and macaque, respectively. Vertical grey box demarcates mid-fetal developmental periods. For all of those plots, the shading around the lines represents the 95% confidence interval. Predicted ages, timeline of human and macaque development and the associated periods are shown below.

Extended Data Fig. 10 Quantification of excitatory and inhibitory postsynaptic puncta.

ad, PD 0 WT and hCbln2 E2 mPFC L2-3 and L5 (BCL11B-immunopositive) immunostained for PSD-95/DLG4 or GPHN. Density of PSD-95/DLG4+ excitatory and GPHN+ inhibitory postsynaptic puncta in PD 0 and PD 60 mPFC, primary somatosensory area (SSp), and primary visual area (VISp) of WT (blue), hCbln2 E2 (orange), and Sox5 cKO mice (green). Two-tailed Student’s test; *P = 0.018; ***P = 4e-4, 1e-4 (b, WT vs. humanized hCbln2 E2 L2-3, and L5, respectively), 1e-4, 7e-5, 4e-4, 2e-4 (d, WT vs. humanized hCbln2 E2 L2-3 and L5, respectively; WT vs. Sox5 cKO L2-3 and L5, respectively); ****P < 1e-5. Error bars: S.E.M.; N = 5 per PD 0 genotype; N = 3 per PD 60 genotype. Scale bar, 25 μm.

Extended Data Fig. 11 Analysis of dendritic spines and dendritic complexity.

ac, Quantification of Sholl analysis and representative images (inset) of Golgi stained dendrites and dendritic spines per 10 μm in layer 2-3 and 5 of mPFC (a), SSp (b) and VISp (c) in WT Cbln2 E2 and humanized hCbln2 E2. Two-way ANOVA with Sidak’s multiple comparison method was applied; *P = 0.04; **P = 1e-3, ****P < 0001; Error bars: S.E.M.; N = 16 (WT Cbln2 E2), and 16 (humanized hCbln2 E2) of PD 60 brains (N = 3). Scale bars: 5 mm. d, e, Quantification and representative images of juxtaposed synaptophysin (SYP) and DLG4/PSD-95 immunolabelled puncta in upper (UL) and deep layer (DL) mPFC of WT Cbln2 E2 and humanized hCbln2 E2. Two-tailed Student’s t-test was applied * P = 0.03; Error bars: S.E.M. (N = 3); Scale bar: 10 mm. f, Electron microscopy (EM) image shows dendrite emitting spine with postsynaptic density (PSD) and spine apparatus (SA) (left) in PD 60 humanized hCbln2 E2 mouse. 3D reconstruction (right) of the dendrite with numerous spines showing spine heads and synaptic contacts (red). Double arrow points to the spine shown in EM image. Thin spines without head (TS) and not innervated thin extensions (arrow) are also detected. Abbreviation: M, mitochondria. Sixteen-representable dendrite fragments were traced in the serial images (see Methods). g, Quantification of percentage of innervated spines in mPFC of WT Cbln2 E2 and humanized hCbln2 E2. Two-tailed Student’s t-test was applied. NS, Not significant. Error bars: S.E.M. (N = 3).

Supplementary information

Supplementary Fig. 1

Full image of ChIP-PCR assays from Figure 2c in PD 0 mouse neocortex using anti-RNA polymerase II (anti-POL2), IgG, and anti-SOX5 and Cbln2 E1 and E2 primers with relevant marker sizes defined.

Reporting Summary

Supplementary Table 1

Comparative analysis of the transcription factor binding sites in CBLN2 E2.

Supplementary Table 2

List and sequence of relevant primers.

Supplementary Table 3

Statistics for all comparisons in Figure 1a.

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Shibata, M., Pattabiraman, K., Muchnik, S.K. et al. Hominini-specific regulation of CBLN2 increases prefrontal spinogenesis. Nature 598, 489–494 (2021). https://doi.org/10.1038/s41586-021-03952-y

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