GoKawiilHuman tissue and ethical approval
Post-mortem human brain specimens were obtained from the Department of Neuroscience at Yale University School of Medicine, the Birth Defects Research Laboratory at the University of Washington, Advanced Bioscience Resources (ABR), the Human Brain Collection Core (HBCC), the Brain and Tissue Bank at the University of Maryland, the MRC–Wellcome Trust Human Developmental Biology Resource at the Institute of Human Genetics, University of Newcastle (UK) and the Human Fetal Tissue Repository at the Albert Einstein College of Medicine (AECOM). All tissue was collected with informed consent from parents or next of kin and under protocols approved by the institutional review boards of Yale University School of Medicine, the National Institutes of Health and the corresponding institutions from which specimens were obtained. Tissue processing complied with NIH ethical guidelines and the principles of the WMA Declaration of Helsinki (https://www.wma.net/policies-post/wma-declaration-of-helsinki/).
Human cortical developmental stages were defined according to the 15-period framework of prenatal brain development (periods 1–15), each corresponding to specific neurodevelopmental milestones38,40,49,50. In this study, we focused on the mid-fetal stage (periods 4–6; PCW13–24)—a developmental window characterized by consolidation of the cortical plate, expansion of the subplate zone, migration of upper-layer projection neurons and ingrowth of thalamocortical afferents, which are closely associated with the establishment of cortical circuitry and regional identity38,40,50,51. On the basis of previous cross-regional transcriptomic analyses demonstrating that cortical areal transcriptional differences are most pronounced during the mid-fetal period21,23,38,40,49, as well as our previous work implicating RA signalling in PFC development during this stage23,24, we restricted our analyses to samples within this window. Within the mid-fetal period, PCW18 was selected as the primary time point, as it lies near the midpoint of this developmental window and captures the key neurodevelopmental processes described above. Owing to ethical and practical constraints associated with the acquisition of primate—particularly human—fetal tissue, sample availability is inherently limited. As a result, minor variability in sampling across datasets is present. Nevertheless, all samples included in this study fall within the defined mid-fetal period and correspond to the relevant neurodevelopmental milestones outlined above. While earlier developmental stages are important for initial cortical patterning, the present study was specifically designed to investigate the establishment and refinement of areal identity, which are most prominently observed during the mid-fetal stage.
Macaque tissue and ethical approval
Rhesus macaque brain samples were collected post-mortem from Yale MacBrain Resource Center (MBRC). All experiments using macaques were carried out in accordance with protocols approved by Yale University’s Committee on Animal Research and NIH guidelines.
Developmental stage correspondence between human and macaque was determined based on cross-species neurodevelopmental timing analyses76, which estimate that human mid-fetal stages (such as PCW18) correspond to macaque developmental stages around PCD79. Owing to the limited availability of macaque fetal samples, an exact stage match was not feasible; therefore, samples closest to this time point (PCD80) were selected for analysis. We also included a PCD149 sample as complementary evidence. As the PCD80 tissue was freshly frozen, the sections were fixed in 4% paraformaldehyde (PFA) for 15 min before IHC. PCD149 whole slabs or whole hemispheres were post-fixed in 4% PFA for 48 h and then cryoprotected in an ascending sucrose gradient (10%, 20%, 30%), with tissue held for 1 week at each step.
Mice used in this study
All experiments involving mice (Mus musculus) were conducted under protocols approved by Yale University’s Institutional Animal Care and Use Committee and in accordance with National Institutes of Health (NIH) guidelines. Mice were housed under controlled environmental conditions (25 °C, 56% relative humidity, 12 h–12 h light–dark cycle) with ad libitum access to food and water. Experimental cohorts included both sexes. The day of vaginal plug detection was designated as embryonic day 0.5 (E0.5), and the day of birth as PD0. The following mouse lines were used: C57BL/6J, Rarb-KO23, Rxrg-KO23, RARE-lacZ (Tg(RARE-Hspa1b/lacZ)12Jrt; Jackson Laboratory) and Neurod6-cre (Nex1-Cre)77. We did not formally calculate sample sizes; we estimated the number of animals needed on the basis of established practices in the field and previous studies using these experimental approaches. For all experiments, the number of animals or biological replicates (n) is indicated either in the figure legend or in the associated Supplementary Table. Groups of animals included specific genotypes and therefore randomization was not applicable here. Before surgery or tissue collection, the animals were given identification numbers that did not contain genotype information; however, visual differences between mutant and control mice precluded true blinding.
Generation of Meis2 flox line
Mice carrying a conditional floxed Meis2 allele were generated by CRISPR–Cas9–mediated gene editing according to previously described methods78,79. Cas9 target (protospacer) sequences in introns 2 and 3 of the Meis2 gene were determined using the MIT CRISPR tool (http://crispr.mit.edu), and the loxP sites flanking exon 3 were inserted (Extended Data Fig. 6a,b). Single-guide RNAs (sgRNAs) targeting these protospacers were transcribed in vitro and purified using the MEGAShortscript kit (Invitrogen) and the MEGAclear kit (Invitrogen), respectively. Single-stranded oligodeoxynucleotide (ssODN) repair templates containing loxP sites were synthesized by IDT Technologies. The floxed allele was generated in two steps: first by introducing the 5′ loxP site, followed by breeding and subsequent targeting of the 3′ loxP site. sgRNA–Cas9 ribonucleoproteins and the corresponding ssODN repair template were electroporated into C57BL/6J (Jackson Laboratory) zygotes79. Embryos were then transferred into the oviducts of pseudopregnant CD-1 foster female mice using standard methods. Founder animals were identified by PCR and sequencing of the targeted loxP sites. Correct targeting and germline transmission of the conditional allele were confirmed by breeding with C57BL/6J mice. Genotyping was performed by PCR using the following primers: forward: 5′-CTCGGCTGATTGAGGGTGTAGTG-3′; reverse, 5′-AGAGACACACGCACGGAGATG-3′.
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