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Regulation of prefrontal patterning and connectivity by retinoic acid

Nature volume 598, pages 483–488 (2021)Cite this article

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Abstract

The prefrontal cortex (PFC) and its connections with the mediodorsal thalamus are crucial for cognitive flexibility and working memory1 and are thought to be altered in disorders such as autism2,3 and schizophrenia4,5. Although developmental mechanisms that govern the regional patterning of the cerebral cortex have been characterized in rodents6,7,8,9, the mechanisms that underlie the development of PFC–mediodorsal thalamus connectivity and the lateral expansion of the PFC with a distinct granular layer 4 in primates10,11 remain unknown. Here we report an anterior (frontal) to posterior (temporal), PFC-enriched gradient of retinoic acid, a signalling molecule that regulates neural development and function12,13,14,15, and we identify genes that are regulated by retinoic acid in the neocortex of humans and macaques at the early and middle stages of fetal development. We observed several potential sources of retinoic acid, including the expression and cortical expansion of retinoic-acid-synthesizing enzymes specifically in primates as compared to mice. Furthermore, retinoic acid signalling is largely confined to the prospective PFC by CYP26B1, a retinoic-acid-catabolizing enzyme, which is upregulated in the prospective motor cortex. Genetic deletions in mice revealed that retinoic acid signalling through the retinoic acid receptors RXRG and RARB, as well as CYP26B1-dependent catabolism, are involved in proper molecular patterning of prefrontal and motor areas, development of PFC–mediodorsal thalamus connectivity, intra-PFC dendritic spinogenesis and expression of the layer 4 marker RORB. Together, these findings show that retinoic acid signalling has a critical role in the development of the PFC and, potentially, in its evolutionary expansion.

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Fig. 1: Predicted RA-signalling-associated genes are upregulated in human mid-fetal prospective frontal areas.
Fig. 2: PFC-enriched anterior–posterior gradient of RA.
Fig. 3: Reduced RA signalling in the PFC of mice lacking Rarb and Rxrg leads to the downregulation of genes involved in synapse and axon development.
Fig. 4: Altered mPFC–MD connectivity in mice lacking Rarb and Rxrg.
Fig. 5: Increased RA signalling promotes mPFC–medial thalamic connectivity.

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Data availability

The mouse RNA-seq data are available at the NCBI GEO under the accession number GSE142851.

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Acknowledgements

We thank S. Bai and T. Nottoli for technical help in the generation of gene-edited mouse lines; F. Hyder and J. Mihailovic for their assistance with MRI diffusion-weighted sequence design and conducting the MRIs; A. Duque for the use of equipment from MacBrainResource (MH113257); J. Rubenstein for sharing reagents; F. Li and M. Onorati for generating human iPS cells used in this study; Y. Gilad and B. Pavlovic for providing chimpanzee iPS cells; and the members of the N.S. laboratory for comments. This work was supported by the NIH (HG010898, MH106874, MH106934, MH110926 and MH116488) and the Simons Foundation Autism Research Institute (SFARI) 572080 (N.S.). The project that gave rise to these results received the support of a fellowship from the 'La Caixa' Foundation (ID 100010434). The fellowship code is LCF/BQ/PI19/11690010. Additional support was provided by the NIH T32 fellowship (MH18268) (K.P.), the National Science Foundation Graduate Research Fellowship Program (S.K.M.), the Ministerio de Ciencia e Innovación, Spain (PID2019-104700GA-I00) (G.S.), the Kavli Foundation and the James S. McDonnell Foundation (N.S.).

Author information

Author notes

  1. These authors contributed equally: Mikihito Shibata, Kartik Pattabiraman

Authors and Affiliations

  1. Department of Neuroscience, Yale School of Medicine, New Haven, CT, USA

    Mikihito Shibata, Kartik Pattabiraman, Belen Lorente-Galdos, David Andrijevic, Suel-Kee Kim, Navjot Kaur, Sydney K. Muchnik, Xiaojun Xing, Gabriel Santpere, Andre M. M. Sousa & Nenad Sestan

  2. Yale Child Study Center, Yale School of Medicine, New Haven, CT, USA

    Kartik Pattabiraman

  3. Department of Genetics, Yale School of Medicine, New Haven, CT, USA

    Sydney K. Muchnik & Nenad Sestan

  4. Yale Genome Editing Center, Yale School of Medicine, New Haven, CT, USA

    Xiaojun Xing & Nenad Sestan

  5. Neurogenomics Group, Research Programme on Biomedical Informatics (GRIB), Hospital del Mar Medical Research Institute (IMIM), DCEXS, Universitat Pompeu Fabra, Barcelona, Spain

    Gabriel Santpere

  6. Waisman Center, University of Wisconsin–Madison, Madison, WI, USA

    Andre M. M. Sousa

  7. Department of Neuroscience, University of Wisconsin–Madison, Madison, WI, USA

    Andre M. M. Sousa

  8. Department of Comparative Medicine, Yale School of Medicine, New Haven, CT, USA

    Nenad Sestan

  9. Department of Psychiatry, Yale School of Medicine, New Haven, CT, USA

    Nenad Sestan

  10. Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale School of Medicine, New Haven, CT, USA

    Nenad Sestan

  11. Kavli Institute for Neuroscience, Yale University, New Haven, CT, USA

    Nenad Sestan

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Contributions

M.S., K.P. and N.S. designed the research. M.S., K.P. and N.K. performed mouse experiments and analysed the data. B.L.-G. and S.K.M. analysed human and mouse transcriptomic datasets. G.S. analysed the enrichment of binding sites for RA receptors. S.-K.K. performed and analysed primate organoid and human neuronal primary culture experiments. M.S. generated the constructs for mutant mice lines. X.X. performed pronuclear injection. D.A. and K.P. analysed mouse imaging data. M.S. and A.M.M.S. performed post-mortem human and macaque tissues experiments. 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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Extended data figures and tables

Extended Data Fig. 1 Workflow for analysis of mid-fetal RNA-seq data and genes upregulated in individual lobes.

a, Human developmental neocortical RNA-seq dataset and workflow for analysis to identify genes upregulated in each cortical lobe. b, Genes upregulated in the frontal, temporal, parietal and occipital lobes in comparison to the other lobes. We identified 190 protein-coding genes that are specifically upregulated, using stringent criteria, in at least one area within a lobe in comparison with areas from other lobes: 125 in the frontal lobe, 46 in the temporal lobe, 17 in the parietal lobe, and 2 in the occipital lobe. The X-axis represents proportion of putative areas in the frontal lobe in which the gene is significantly upregulated according to RNentropy. The Y-axis represents proportion of areas in the other lobes in which the gene is significantly downregulated according to RNentropy. Upregulated genes, the ones delimited by dashed red lines, are labelled. c, d, Gene loadings of PC1 from PCA of protein-coding genes that are specifically enriched in one of the four lobes of the mid-fetal human (c) and macaque (d) cortex. Colours represent the cortical lobe where the gene was found to be specifically upregulated. For reproducibility information, see Methods.

Extended Data Fig. 2 Analysis of genes upregulated in individual cortical lobes during other developmental periods.

a, Analysis of upregulated genes during window 2 (early fetal period, PCW 12-13 specimens in the BrainSpan dataset17). See Fig. 1a for further explanation. b, Number of upregulated genes during late prenatal, and early postnatal stages in individual lobes are significantly reduced compared to earlier stages. c, d, Analysis of upregulated genes during window 8 (postnatal year, PY 13-19) and W9 (PY 21-64). See Fig. 1a for further explanation. For reproducibility information, see Methods.

Extended Data Fig. 3 Extended RA-specific GO analysis and spatiotemporal expression of select genes upregulated in the mid-fetal frontal lobe.

a, Analysis for statistically significant enrichment of upregulated genes during developmental and adult stages for GO terms associated with RA. X-axis represents windows analysed, which are defined at the bottom of the figure. b, Spatiotemporal expression of select genes upregulated in the mid-fetal frontal lobe from Fig. 1 in sixteen neocortical areas across human (red) and macaque (blue) development using BrainSpan (brainspan.org) and PsychENCODE (evolution.psychencode.org) RNA-seq data17,19. Thick full lines represent four PFC areas, thick dotted line represents the primary motor cortex (M1C) and thin dotted lines represent the other non-frontal neocortical areas. Vertical grey box demarcates mid-fetal periods analysed in Fig. 1. Timeline of human and macaque development and the associated periods designed by Kang et al39. shown below. Predicted ages were calculated using the TranscriptomeAge algorithm19, which aligns our earliest macaque samples (PCD 60) with human early mid-fetal samples. Distinct global patterns of spatiotemporal expression were observed. For example, precocious expression in the frontal lobe/PFC followed by broad expression in all eleven neocortical areas (e.g. CBLN2, BDNF), transient enrichment in the frontal lobe/PFC (e.g. WNT11, PCDH17) and downregulation in non-PFC areas during mid-fetal development (e.g. MEIS2).

Extended Data Fig. 4 Expression of RA-synthesizing enzymes in the developing human and macaque cortex.

a, Spatiotemporal expression of genes encoding RA synthesizing enzymes, ALDH1A1, 2 and 3, in eleven neocortical areas of human and macaque during prenatal and postnatal development using BrainSpan (brainspan.org) and PsychENCODE (evolution.psychencode.org) RNA-seq data17,19. Red and blue lines indicate human and macaque, respectively, and dotted lines represent the non-PFC expression in the PFC plot. Vertical grey box demarcates mid-fetal developmental periods. Predicted ages, timeline of human and macaque development, and the associated periods are shown below19,63. b, Heat map of normalized (z-score) microarray signals computed for genes encoding RA synthesizing enzymes from the BrainSpan human prenatal laser microdissection microarray data63 (brainspan.org). Left columns represent gene name and specific probe. Each column represents regions of the brain labelled above the heat maps. Darker reds represent high expression levels. c, Anteroposterior visual representation of human ALDH1A1 and ALDH1A3 expression at PCW 16 and 15 respectively from BrainSpan atlas58. ALDH1A1, 2 and 3 expression in mid-fetal human (PCW 19, 19, 20) (d, left) and macaque (PCD 80, 80, 110) (e, left) meninges. Immunostaining for ALDH1A1 and TH in human PCW 12 and 22 brains (d, right), and in macaque PCD 76 brain (e, right). White arrowheads, and open arrowheads indicate ALDH1A1+;TH+ and ALDH1A1+;TH- axons in the subplate, respectively. NCX, neocortex; PU, putamen; NAC, nucleus accumbens; CA, CA subfields of hippocampus. Errors bars: S.D. N = 3. f, Anterior to posterior expression of ALDH1A3 mRNA in human (PCW 21), macaque (PCD 140) and mouse (PD 0) brain. N = 2 for human and macaque, N = 3 for mouse. g, Western blot using ALDH1A3 antibody in human (PCW 20), macaque (PCD 114) and mouse (PD 0) frontal cortex areas. In macaque and human, there are two bands that are likely to represent ALDH1A3 (56 kDa) and ALDH1A1 (54 kDa). Experiments were repeated at least two times for each animal species. See Extended Data Fig. 6a for schemas of frontal areas.

Extended Data Fig. 5 Expression of genes encoding RA-synthesizing enzymes in the developing mouse cortex.

Expression of Aldh1a1, Aldh1a2 and Aldh1a3 at PCD 11.5, 18.5 and PD 4 (a) and Rara at PCD 15.5 and PD 4 (b) from the Allen Developing Mouse Brain Atlas58 (developingmouse.brain-map.org). c, Expression of RA related genes in the PCD 18.5 choroid plexus (CP). Blue bars represent lateral ventricle CP and orange bars represent fourth ventricle CP. Data from Lun et al64. d, Analysis of Rarb-gfp mouse line from the GENSAT project59 (gensat.org) at PD 7 revealed GFP expression in upper (open arrows) and deep layer (solid arrows) pyramidal neurons in the mPFC at PD 7. Scale bars: 1 mm. FEZ, Frontonasal ectodermal zone; ChPl, Choroid plexus; RMS, Rostral migratory stream; SN, Substantia nigra; VTA, Ventral tegmental area; mPFC, Medial prefrontal cortex; ACA, anterior cingulate area.

Extended Data Fig. 6 Expression of RA-synthesizing enzymes in the developing and adult cortex.

a, Representative images of ALDH1A1 and ALDH1A3 in situ hybridization of human (PCW 21; N = 2), macaque (PCD 110 and 140; N = 1 each), and mouse (PD 0; N = 3) PFC. Red, black, and open arrowheads indicate ALDH1A1-expressing subplate neurons (insets), astrocytes and meninges, respectively. Scale bars: 200 μm (mouse); 2 mm (human and macaque); 100 μm (mouse, lower panel); 500 μm (human and macaque, lower panel). b, ALDH1A1 immunofluorescence (green) and immunohistochemistry (brown). Human: left, solid and open arrowheads indicate TH+;ALDH1A1+ and TH+;ALDH1A1- axons, respectively; middle, solid and open arrowheads indicate subplate neurons and astrocytes, respectively; right, arrowheads indicate GFAP+;ALDH1A1+ astrocytes. Macaque: left, solid and open arrowheads indicate TH+;ALDH1A1+ and TH+;ALDH1A1- axons, respectively; middle, solid and open arrowheads indicate putative excitatory NRGN+;NR4A2+;ALDH1A1+ and inhibitory GAD1+;ALDH1A1- subplate neurons, respectively; right, arrowheads indicate GFAP+;ALDH1A1+ astrocytes. Mouse: left, solid and open arrowheads indicate TH+;ALDH1A1+ and TH+;ALDH1A1- mPFC axons (inset), respectively; middle, TH+ (red) and ALDH1A1+ (green) midbrain neurons and axons in striatum (STR), lateral septal nucleus (LSR) and cortex (CTX); right, GFAP+;ALDH1A1- astrocytes. N = 3. Scale bars: 20 μm (human, macaque, and mouse left and middle panels); 1 mm (mouse right panel). For reproducibility information, see Methods.

Extended Data Fig. 7 Expression of genes encoding RA-degrading enzymes in the developing human and macaque cortex.

a, Expression of RA degrading enzymes in individual regions of the cerebral cortex of human and macaque during development. Red and blue lines indicate human and macaque, respectively, and dotted lines represent the non-PFC expression in the PFC plot and vice versa. Vertical grey box demarcates mid-fetal developmental periods. Predicted ages, timeline of human and macaque development, and the associated periods are shown below19,39. * Data for CYP26A1 was not present in Zhu et al19. and the data analysed individually. The expression level for human and macaque were not normalized and can’t be directly compared. b, Heat map of normalized (z-score) microarray signals computed for genes encoding RA degrading enzymes from the BrainSpan human prenatal laser microdissection microarray data63 (brainspan.org). Left column represents gene and specific probe. Rows represent regions of the brain. Darker reds represent high expression levels. c, Anteroposterior visual representation of human CYP26A1 and CYP26B1 expression at PCW 15 respectively from the BrainSpan atlas.

Extended Data Fig. 8 Expression of genes encoding RA receptors in the developing human and macaque cortex.

a, Expression of genes encoding RA receptors in individual regions of the cerebral cortex of human and macaque during prenatal and postnatal development. Red and blue lines indicate human and macaque, respectively, and dotted lines represent the non-PFC expression in the PFC plot and vice versa. Vertical grey box demarcates mid-fetal developmental periods. Predicted ages, timeline of human and macaque development, and the associated periods are shown below19,39. * Data for RXRB was not present in Zhu et al19 and the data were analysed individually. The expression level for human and macaque were not normalized and cannot be compared. b, Heat map of normalized (z-score) microarray signals computed for genes encoding RA receptors from the BrainSpan human prenatal laser microdissection microarray data63 (brainspan.org). Left column represents gene and specific probe. Rows represent regions of the brain. Darker reds represent high expression levels.

Extended Data Fig. 9 RARB and RXRB are expressed in a moderate anterior-to-posterior gradient in the developing neocortex.

a, Quantitative PCR analysis of Rara, b, g and Rxra, b, g transcripts in the anterior and posterior half of mouse cortex at PD 0. Two-tailed Student’s t-test: ***P = 4e-4, 1e-4; N = 3 per condition; Errors bars: S.E.M. b, Quantitative PCR analysis of Rarb and Rxrg transcripts in four sections dissected out of the cortical plate in anterior-posterior direction. Both genes showed an anterior-posterior gradient in expression level. Two-tailed Student’s t-test; N = 3 per condition; Errors bars: S.E.M. c, Expression of Rarb and Rxrg in mouse (PD 0), macaque (PCD 140) and human (PCW 21) brains by in situ hybridization. Higher magnification images of the regions of anterior cortex. Rarb and Rxrg transcripts are upregulated in the anterior part of the cortex in all three. Scale bars, 200 μm (mouse); 2 mm (human); 500 μm (human, higher magnification). N = 2 for human and macaque, N = 3 for mouse. d, Strategies for the generation of Rarb and Rxrg KO mice using CRISPR-Cas9 technique41.

Extended Data Fig. 10 RA signal in the neonatal mouse forebrain.

a, β-galactosidase histochemical staining of more posterior regions of Rarb+/+; Rxrg+/+ (WT); RARE-lacZ and Rarb-/-; Rxrg-/- (dKO); RARE-lacZ mouse brains at PD 0. Signal intensity in the boxed area (ACA, SSp, RSPA, STR, HIP and VP) was quantified. Note the reduced activity in anteromedial structures including ACA and RSPA (RSP). There is also reduced expression in HIP and lateral STR, but not the thalamus. Two-tailed Student’s t-test; *P = 9e-3, **P = 7e-4; 3e-3 (from left), ***P = 2e-4, ****P = 2e-7; 1e-5; 4e-5; 5e-6; 6e-6 (from left). N = 3 per genotype: Errors bars: S.E.M.; Scale bars, 200 μm. b, Cbln2 and Meis2 expression in PD 0 WT and dKO mutant brain by in situ hybridization at PD 0. Note that Cbln2 and Meis2 expression in mPFC was decreased in dKO. Scale bar, 200 μm. N = 3 per genotype. ACA, Anterior cingulate area; CP, caudoputamen; HIP, Hippocampus; RSPA/RSP, Retrosplenial area; STR, striatum; VP, Ventroposterior thalamus.

Extended Data Fig. 11 RA regulates CBLN2, MEIS2 and DLG4 (PSD95) expression in human and chimpanzee cerebral organoids.

a, Expression of cortical neural stem/progenitor markers (PAX6 and SOX2), cortical cell type-specific markers (BCL11B, SATB2 and TBR1) and a pan-neuronal marker (MAP2) across the differentiation times show the dorsal cortical identity of the organoids derived from human and chimpanzee induced pluripotent stem cells. Scale bars, 50 μm. Each experiment used with 3-5 replicate organoids per times and conditions. b, In situ hybridization for CBLN2 and MEIS2, immunostaining for PSD95/DLG4, and DAPI nucleic acid (nuclear) staining in human and chimpanzee day 135 organoids exposed to low or high concentration RA-soaked bead for 48 h. Scale bars, 500 μm. One experiment has done with 3-5 replicate organoids per times and conditions. c, Proportion of TuJ1 immuno-positive neurons in day 135 cerebral organoids was similar across conditions. The ratio of total number of PSD95+ synaptic puncta to total number of TuJ1+ cells in the day 135 cerebral organoids was significantly increased in RA-soaked bead applied conditions. Two-tailed t-test, compared to the condition without bead (Control, Ctrl), Errors bars: S.E.M. ***P = 5e-4, ****P < 1e-4. N=5 (multiple sections for each organoid) per condition.

Extended Data Fig. 12 Additional analysis of RNA-seq experiments.

a, Number of upregulated genes between PD 0 WT and dKO mice per region and phenotype, as well as combinations of regions and phenotypes. b, Gene loadings of the first principal component from PCA in Fig. 3. Colours represent the frontal cortex region where the gene was found to be upregulated. c, Cellular component GO terms associated with the total list of 4,768 DEx genes found, showing their z-score and nominal P values. Z-score represents the proportion of upregulated versus downregulated genes in the list of DEx genes associated to each GO term (i.e. z-score = (#up - #down) / sqrt (#all DEx associated to the GO term)). Dark blue: z-score <−5; light blue: z-score (−5,0]; orange: z-score >0. Size of the bubbles are proportional to the total number of DEx genes associated to the given GO term. d, Cellular component GO terms associated with DEx genes found in all three frontal cortex regions, and DEx genes unique to each region (mPFC, OFC, MOs) and their unadjusted p value. e, Enrichment of RA related genes (green), genes upregulated in individual lobes of the mid-fetal human cortex based on Fig. 1 (purple), and psychiatric disease related genes in up- and downregulated genes (pink). DEx genes are separated by genes that are DEx only in the given region (mPFC, OFC, MOs), genes that are DEx in the two given regions (mPFC + OFC, mPFC + MOs, OFC + MOs), genes that are DEx in all three regions (mPFC + OFC + MOs). Circles plotted for significant enrichments (P value <0.05), in darker colour, significance is considering the adjusted p value. Diameter of circle is associated with odds ratio per legend.

Extended Data Fig. 13 Altered synaptic density and axonal projections in Rxrg and Rarb dKO mice.

a, Example of downregulated genes between PD 0 Rarb+/+; Rxrg+/+ (WT) and Rarb-/-; Rxrg-/- (dKO) that displayed an anterior to posterior gradient in PCD 18 mouse embryo (images are from the Allen Developing Mouse Brain Atlas; developingmouse.brain-map.org/58). Scale bar: 1 mm. b, Immunostaining for PSD95/DLG4 in the cortical subregions (mPFC, MOs, OFC, MOp, and SSp) of WT and dKO brain at PD 0. Each region as shown in Fig. 2c. Scale bar: 25 μm. N = 3 per genotype. c, Quantification and representative images of co-localized synaptophysin (SYP) and PSD95 puncta in PD 0 WT and dKO mPFC. Two-tailed Student’s t-test; *P = 0.01; N = 3 per genotype; Errors bars: S.E.M.; Scale bar: 10 μm. d, e, DiI placement in mPFC (c) and medial thalamus (d) with tracing data in WT and dKO brain at P21. Additional two replicates of experiment shown in Fig. 4d, e are shown. Asterisks: DiI crystal placement. Scale bar: 1 mm.

Extended Data Fig. 14 Analysis of axonal inputs to the mPFC and dendritic spines in Rarb and Rxrg dKO mice.

Anteroposterior series of PD 30 coronal sections of representative WT (a) and dKO (b) with retrograde viral tracer AAVrg-Cag-Gfp injected into mPFC (green asterisk). Sections were inverted and converted to greyscale. Black regions indicate the presence of viral tracer. High magnification representative images of labelled regions (c) and quantification of labelling using 0-3 scale (d) (N = 4 for each genotype; see Methods section for more details). e, Quantification of Sholl analysis (Two-way ANOVA with Sidak’s multiple comparison method; P = .440- >0.999); Error bars: S.E.M.) and dendritic spines of upper layer neurons in the contralateral mPFC in WT (blue) and dKO (orange) (N = 4 for each genotype). Two-way ANOVA with Sidak’s multiple comparison method was applied; *P = 0.09; ****P < 1e-4; Error bars: S.E.M. Inset shows representative images of dendritic spines. Scale bar: 5 μm. ACAd, Dorsal anterior cingulate area; AI, Anterior Insula; BLA, Basolateral amygdala; vHPF, ventral hippocampal fields; CLA, claustrum; cl mPFC, Contralateral medial prefrontal cortex; ILA, Infralimbic area; LHA, Lateral habenula; MD, The mediodorsal nucleus of the thalamus; MOs/p, The secondary and primary motor areas. PER, Perirhinal cortex PIR, Piriform cortex PL, Prelimbic area; SSp, Primary somatosensory area; STN, Subthalamic nucleus; VTA, Ventral tegmental area.

Extended Data Fig. 15 RA regulates CBLN2, MEIS2, and DLG4 (PSD95) expression and synaptogenesis in human fetal neocortical neurons.

a, Schematics of human cortical neuron differentiation and modulation of RA signalling. From PCW 8 neocortical tissue, cortical neural stem cells were isolated and expanded with FGF2 for 20 days. Neural differentiation was initiated by the mitogen withdrawal. After 14 days of differentiation, cells were treated with varying doses of RA or RA receptor inhibitor (RARi, AGN193109) for another 14 days. From PCW 20 or 23 neocortical tissue, cortical neural progenitors and neurons were isolated and cultured without FGF2. After 16 days of culture, cells were treated with RA or RARi for another 14 days. Gene expression of cell type markers (b), RA receptors (c) RA regulated genes CBLN2 and MEIS2 (d) in PCW 8 cortical cells after 28 days of differentiation, measured by ddPCR. One way ANOVA and multiple comparison, compared to vehicle (0.01% DMSO)-treated control. Errors bars: S.E.M. *P < 0.05 (RXRA expression, RA1.0, P = 0.0192, RARi5, P = 0.0277; CBLN2 expression, RARi10, P = 0.0135; MEIS2 expression, RA1.0, P = 0.0456), **P < 0.01 (RARA expression, RARi10, P = 0.0039; MEIS2 expression, RARi5, P = 0.0057, PARi10, P = 0.0028), ***P < 0.001 (MEIS2 expression, RA0.5, P = 0.0003), ****P<0.0001. N=3 experimental replicates per condition. e, TUBB3/TUJ1 and DLG4/PSD95 expression in RA or RARi-treated PCW 8 cortical cells. Scale bar: 50 μm. f, DLG4/PSD95 transcript in PCW 8 cortical cells was significantly increased in RA 1 μM condition quantified by ddPCR. Two-tailed t-test, compared to the vehicle treatment. Errors bars: S.E.M. **P = 0.0083. N= 8 fields per condition. g, The ratio of total DLG4/PSD95+ puncta volume to total TUJ1+ volume in PCW 8 cortical cells was increased in the RA condition and reduced in the RARi condition. Two-tailed t-test, compared to the vehicle treatment. Errors bars: S.E.M. *P = 0.0152, ***P = 0.0001. h, MAP2, synaptophysin (SYP) and DLG4/PSD95 expression in RA or RARi-treated PCW 20 and 23 cortical cells. Scale bar: 50 μm. i, The ratio of total DLG4/PSD95+ puncta volume to total MAP2+ volume in PCW 20 and 23 cortical cells was increased in RA conditions and reduced in RARi conditions. Two-tailed t-test, compared to the vehicle treatment. Errors bars: S.E.M. *P <0.05 (PCW 20-RA2, P = 0.0221; PCW 20 -RARi5, P = 0.0193; PCW 23 -RA0.5, P = 0.0192), **P <0.01 (PCW 20-RARi10, P = 0.0018; PCW 23-RA1, P = 0.0081), ***P = 0.0004. N = 8 fields per condition. j, The ratio of total number of SYP+ and DLG4/PSD95+ colocalized synaptic puncta to total MAP2+ volume in PCW 20 and 23 neocortical cells was increased in RA conditions and reduced in RARi conditions. Two-tailed t-test, compared to the vehicle treatment. Errors bars: S.E.M. *P (PCW 20 -RA0.5, P = 0.0271; PCW 20-RA2, P = 0.0159; PCW 23-RA1, P = 0.0289; PCW 23-RA2, P = 0.0447), ***P = 0.0004, ****P < 0.0001. N= 8 fields per condition.

Extended Data Fig. 16 Analysis of axonal projections and cell death in Rarb and Rxrg dKO mice.

a, Four scalar indexes which describe microstructural integrity do not differ in the four major white-matter tracts (corpus callosum, anterior commissure, left and right internal capsules) between WT and dKO mice. FA Comparisons: Corpus callosum (two-tailed unpaired t-test, P = 0.4); Anterior cingulate (two tailed mann-whitney test, P = 0.2); Internal Capsule left (two-tailed unpaired t-test, P = 0.07); Internal Capsule right (two-tailed unpaired t test, P = 0.8). ADC Comparisons: Corpus callosum (two-tailed unpaired t-test, p value 0.1); Anterior cingulate (two-tailed unpaired t-test, p value 0.1); Internal Capsule left (two-tailed unpaired t-test, p value 0.9); Internal Capsule right (two-tailed unpaired t-test, P = 0.3). RD Comparisons: Corpus callosum (two-tailed unpaired t-test, P = 0.1); Anterior cingulate (two-tailed unpaired t-test, P = 0.1); Internal Capsule left (two tailed mann-whitney test, P = 0.1); Internal Capsule right (two-tailed unpaired t-test, P = 0.3). AD Comparisons: Corpus callosum (two-tailed unpaired t-test, P = 0.08); Anterior cingulate (two-tailed unpaired t-test, P = 0.1); Internal Capsule left (two-tailed unpaired t-test, P = 0.4); Internal Capsule right (two-tailed unpaired t-test, P = 0.2). N=5; Errors bars: S.E.M. b, Number of streamlines of MOp-thalamus and corticocortical tracts, did not differ between WT and dKO. Two-tailed unpaired t-test for MOp-MOp, MOp-Th, and AUDp-th (P = 0.1, 0.07, and 0.6, respectively). Two-tailed Mann-Whitney test for ACA-Th (P = 0.3). N = 5 per genotype; Errors bars: S.E.M. c, Barrel formation was examined by Nissl staining at PD 5 in WT and dKO. N = 3 per genotype. d, Representative images and quantification of corticospinal tract (CST) width using L1CAM expression at PD 5 in WT and dKO. The width of the corticospinal tract (shown in brackets) is slightly increased in dKO. Two-tailed Student’s t-test: *P = 0.02; N = 5 per genotype; Errors bars: S.E.M. e, Representative images and quantification of of apoptotic cells in the mPFC detected by cleaved caspase3 (actCASP3) between WT and dKO. Two-tailed Student’s t-test: WT vs. dKO; NS; N = 5 per genotype; Errors bars: S.E.M.; Scale bars: 500 μm; 200 μm (b,f); 1 mm (c); 100 μm (d). f, Representative images and quantification of tyrosine hydroxylase (TH) immunolabelled axons in WT and dKO mouse frontal cortex at PD 5 (N = 4). Rectangles represent regions analysed. Clockwise starting from left-most: mPFC, MOs, OFC. Errors bars: S.E.M.

Extended Data Fig. 17 Analysis of neocortical layers and tissue volume in Rarb and Rxrg dKO mice.

a, Representative images and quantification of total volume of the brain (left), cerebral neocortex (middle), anterior one-third of the neocortex (right) of WT littermates and Rarb;Rxrg dKO at PD 5. Two-tailed unpaired t-test for total volume of the brain and anterior one-third of the neocortex. Two-tailed Mann-Whitney test for volume of cerebral neocortex. P = 0.3, 0.1, 0.6. N = 5. Errors bars: S.E.M. b, Number of immune-positive cells expressing cortical layer markers POU3F2/BRN2, CUX1, RORB, BCL11B/CTIP2, TBR1 in WT and dKO mouse mPFC, MOs, and primary visual cortex (VISp) at PD 5 (N = 3). (Unpaired t-test: WT vs. dKO *P = 0.03; Errors bars: S.E.M.). RORB expression in mouse PFC was too faint to quantify. Below each graph is a representative image of each marker. Scale bar: 200 μm.

Extended Data Fig. 18 RA signal in posterior cortical regions of Cyp26b1 KO mice.

a, Strategy for the generation of Cyp26b1-/- (Cyp26b1 KO) mice using CRISPR-Cas9 gene editing technique41. b, Cyp26b1 expression in PD 0 mice cortex by in situ hybridization. The colorimetric staining was purposefully extended compared to the experiment in Fig. 5a to better visualize low expressing locations. c, β-Galactosidase staining of more posterior regions of control Cyp26b1+/+; RARE-lacZ (Ctrl) and Cyp26b1-/-; RARE-lacZ (KO) mouse brains at PCD 18. Scale bar: 500 μm. Intensity of signal in the boxed areas (ACA, MO, SSp) was quantified. Increase in RA signalling in Cyp26b1 KO brains is less significant in posterior regions. Two-tailed Student’s t-test: Ctrl vs. Cyp26b1 KO: *P = 0.01; 0.04; 9e-3; 7e-3; 6e-3; 0.02; 0.02; **P = 4e-3; 1e-3; 4e-3; 4e-3, N = 3 per genotype; Errors bars: S.E.M.; Scale bars: 200 μm.

Extended Data Fig. 19 Rorb expression and medial thalamocortical projections in neonatal Cyp26b1 KO mice.

a, Additional two replicates of Rorb expression in WT and Cyp26b1 KO brains from Fig. 5f are shown. Scale bar: 100 μm. b, DiI was placed in the medial thalamus of WT and Cyp26b1 KO brains, and signal was detected in the PFC. Additional two replicates of experiment in Fig. 5d are shown. N = 3 per genotype and condition. Arrowheads: Thalamocortical innervation of the medial and dorsolateral frontal cortex. Asterisks: DiI crystals placed. Scale bar: 400 μm. c, DiI was placed in the frontal motor cortex of WT and Cyp26b1 KO brains, and signal was detected in the medial thalamus. Additional two replicates of experiment in Fig. 5e are shown. N = 3 per genotype and condition. Of note, owing to restriction related to Covid-19, PD 0 brains were left 2 months in cold room after 3 weeks in 37 degree. Scale bar: 400 μm.

Extended Data Fig. 20 Ectopic RA signalling leads to enlargement of the lateral region of the frontal cortex and upregulation of Rorb.

a, Representative image and quantification of the ratio of brain width at anterior and posterior cortex to total brain length at PCD 18 in Cyp26b1 KO brain compared WT. Two-tailed Student’s t-test: WT vs. Cyp26b1 KO: ***P = 3e-4; N = 4 per genotype; Errors bars: S.E.M. b, Nissl staining reveals that the cortical wall and cortical plate are grossly normal when analysed in the mPFC and SSp of Cyp26b1 KO. N = 3 per genotype. c, Representative image and quantification of Rorb expression after electroporation of either control pCAG-IRES-Gfp or pCAG-Aldh1a3-IRES-Gfp expression vector plasmid in the dorso-lateral fronto-parietal wall at PCD 14. Brains were dissected out at PD 5. Boxed region represents region of higher magnification to the right. GFP expression as a marker of misexpressing cells are shown in lower panels. Rorb signal intensity in the boxed area in the cortex was quantified. Two-tailed Student’s t-test: ***P = 2e-4; N = 4 per genotype; Errors bars: S.E.M.; Scale bars: 500 μm (a); 200 μm (b); 1 mm (c); 40 μm and 500 μm (d).

Supplementary information

Supplementary Text

This file contains additional results and discussion.

Reporting summary

Supplementary Figure 1

Original picture of western blot gel from Extended Data 4g.

Supplementary Table 1

Gene ontogeny of mid-fetal frontal lobe enriched genes

Supplementary Table 2

Annotation of mid-fetal prospective PFC and M1C enriched genes

Supplementary Table 3

List of differentially expressed genes from wild-type and Rarb;Rxrg dKO PD 0 frontal cortex. obtained using RNentropy72. In brief, the analysis is done in two steps: (i) a global sample specificity test is computed based on their specificity with a background distribution, adjusting their P values with a Benjamini–Hochberg procedure; (ii) the expression in each sample of the significant genes that passed the default global threshold in step (i) are then compared with the expression in the remaining samples with a local sample specificity test, obtaining unadjusted P values. In this table, those results are summarized per phenotype.

Supplementary Table 4

List and annotation of PD 0 mouse differentially expressed or human PFC enriched genes related to axon development

Supplementary Table 5

List of relevant primers and sgRNA

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Shibata, M., Pattabiraman, K., Lorente-Galdos, B. et al. Regulation of prefrontal patterning and connectivity by retinoic acid. Nature 598, 483–488 (2021). https://doi.org/10.1038/s41586-021-03953-x

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