Adaptive evolution of gene regulatory networks in mammalian neocortex | Nature

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Subjects

- Cell type diversity

- Evolutionary developmental biology

- Molecular neuroscience

Abstract

Mammals have evolved a more complex brain, exemplified by the transformation of the single-layer dorsal cortex of excitatory projection neurons (ExNs) in ancestors into a multilayered cerebral neocortex1,2,3,4 enriched with diverse intratelencephalic and extratelencephalic ExN subtypes5,6,7, thereby establishing specialized projection systems that enhance brain connectivity and functionality5,6,7,8. This is in contrast to modern reptiles and birds with single-layered or pseudolayered columnar organization of ExNs4,9,10,11,12. However, the mechanisms underlying these mammalian-specific adaptations remain elusive. By comparing the landscape of gene expression and putative cis-regulatory elements (CREs) in mouse ExN subtypes and through cross-species examination, we identified mammalian-specific CREs, including a subset bound by the transcription factor ZBTB18 (also RP58, ZFP238 or ZNF238) and associated with genes defining intratelencephalic and extratelencephalic subtypes and connectivity, which have been implicated in intellectual disability and autism. Deletion of Zbtb18 in mouse ExNs dysregulated target gene expression, reduced molecular diversity, diminished cortico-spinal and callosal projections and increased intrahemispheric cortico-cortical association projections to the prefrontal cortex, thereby resembling non-mammalian brain. ZBTB18 binding motifs are highly enriched in callosally projecting intratelencephalic-biased putative CREs and show higher conservation specifically in mammals. This study uncovers critical components and mammalian-specific evolutionary adaptations within a regulatory node essential for neocortical ExN identity and connectivity.

Main

Previous studies identified transcription factors (TFs) that guide subtype specification, laminar positioning and connectivity of mammalian neocortical ExNs5,6,7; however, the evolutionary adaptations and precise molecular mechanisms remain elusive. Here we uncovered gene regulatory subcircuits, particularly involving ZBTB18–CRE interactions that govern key features of neocortical ExNs that have undergone modifications in the mammalian lineage.

Subtype-specific CREs and TFs in neocortical ExNs

To characterize CREs and TFs for neocortical ExNs, we used Arpp21–Gfp or Fezf2–Gfp transgenic mice and enriched GFP-expressing neocortical upper layer (L2–4) intratelencephalic (IT) neurons or deep layer (L5–6) predominantly extratelencephalic (ET) neurons, respectively (Fig. 1a and Supplementary Fig. 1), from neonatal mice (postnatal day (PD) 0), an age at which neocortical ExN identity and connectivity are established. Cells were processed for RNA sequencing (RNA-seq) and chromatin immunoprecipitation (ChIP) assays with DNA sequencing (ChIP–seq) for H3K27ac13,14,15,16,17 (Fig. 1b).

Fig. 1: Mammalian-specific changes in the ZBTB18-associated CREs and TF expression in neocortical ExNs.

a, Immunolabelling for Arpp21–Gfp+ IT (IT neurons onwards) and Fezf2–Gfp+ ET (ET neurons onwards) neurons and BCL11B in mouse PD 0 neocortex. b, Schematic for isolation and processing of Arpp21–Gfp-positive and Fezf2–Gfp-positive cells from neocortex for RNA-seq and H3K27ac ChIP–seq. c, TFBS for 69 TFs enriched among both IT-biased H3K27ac peaks (red circle) and H3K27ac peaks near IT-biased genes (blue circle) compared with ET-biased peaks and genes, respectively. TFBS enrichments were tested using Fisher’s exact test. Significant TF motifs had a Benjamini–Hochberg-corrected P value < 0.05 and odds ratio > 1. For RNA-seq, n = 3 biological replicates per condition. For ChIP–seq, n = 2 biological replicates per condition. d, Schematic showing two dorsal pallial regions (H and M) microdissected from E17 chicken embryo. e, Venn diagram showing the co-occurrence of peaks derived from ZBTB18-HA ChIP–seq and H3K27ac peaks preferably found in IT and ET neurons and in chicken dorsal pallium. X are peaks in both IT and ET neurons excluded from analysis. f, Heat map showing pairwise alignment distances among vertebrates for six putative CREs overlapping ZBTB18 ChIP–seq peaks linked to IT neurons and axon guidance. The left grid indicates ChIP–seq peak identification method. Columns on the right show if the regions (H3K27ac or ZBTB18 peaks) are orthologous and active in the chicken embryonic dorsal pallium. Grey columns mark non-orthologous regions; red and black columns show the presence or absence of H3K27ac peaks in orthologous regions. Distances are relative to mice. g, Dot plot of Zbtb18-expressing neurons in the dorsal pallium showing cell percentages and co-expression of genes with the ZBTB18 binding motif in IT neurons across mammalian (mouse) and non-mammalian species (chicken, lizard and turtle). h,i, Coronal sections of chicken (h) and mouse (i) brains showing co-localization of BCL11B and SATB2 with ZBTB18. j, Bar plots showing the co-localization per