(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . A single-cell spatiotemporal transcriptomic atlas of mouse prefrontal cortex maps dynamics of intratelencephalic neurons during postnatal development [1] ['Hu Zheng', 'State Key Laboratory Of Agricultural Microbiology', 'Huazhong Agricultural University', 'Wuhan', 'College Of Veterinary Medicine', 'Keji Yan', 'Xiaojuan Gou', 'Zhongchao Wang', 'Liyao Yang', 'Yayu Huang'] Date: 2026-01 In early postnatal brain, the prefrontal cortex (PFC) remains immature and highly plastic, particularly for the intratelencephalic (IT) neurons. However, the spatiotemporal molecular and cellular dynamics of PFC during this period remain poorly characterized. Here, we performed spatiotemporal single-cell RNA analysis on mouse PFC during different postnatal time points and systematically delineated the molecular and cellular dynamics of mouse PFC during early postnatal development, among which IT neurons exhibit most dramatic alterations. Based on these comprehensive spatiotemporal atlases of PFC, we deciphered the time-specific molecular and cellular characteristics during the maturation process of IT neurons in PFC, particularly the dynamic expression programs of genes regulating axon development and synaptic formation, and the risk genes of neurological developmental diseases. Furthermore, we revealed the dynamic neuron-glia interaction patterns and the underlying signaling pathways during early postnatal period. Our study provided a comprehensive resource and important insights for PFC development and PFC-associated neurological diseases. Funding: This work was supported by the National Natural Science Foundation of China ( https://www.nsfc.gov.cn/) (32221005 to G.C., 32171022 to JX.D., 31900746 to LQ.S., 32401246 to G.C.), the China Postdoctoral Science Foundation ( https://www.chinapostdoctor.org.cn/) (2019M660182 to LQ.S.), the Special Funds Project for Strategic Emerging Industries of Shenzhen Municipal Development and Reform Commission ( https://fgw.sz.gov.cn/) (XMHT20240215002 to G.C.), the Key Areas Special Projects for Ordinary Higher Education Institutions in Guangdong Province ( https://edu.gd.gov.cn/) (2024ZDZX2010 to G.C.), and the Yunnan Provincial Department of Science and Technology Science and Technology Program Project ( https://kjt.yn.gov.cn/) (202503AP140014 to G.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability: The main data supporting the results in this study are available within the paper and its Supporting information files. The raw scRNAseq data are available from GEO (GSE298260). The raw stereo-seq data are available from https://doi.org/10.12412/BSDC.1699433096.20001 . The ISH data are available from https://mouse.brain-map.org/ . The processed data ready for exploration can be accessed and downloaded via our interactive browsers at https://huggingface.co/spaces/TigerZheng/PFCdev-web ( S7 Fig ). All data were analyzed with standard programs and packages. The codes were freely accessible via Zenodo at https://doi.org/10.5281/zenodo.17964214 . Copyright: © 2026 Zheng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. To delineate the molecular and cellular trajectories underlying postnatal development of PFC neurons and investigate the mechanisms governing early neural circuit assembly, we performed high-throughput single-cell RNA sequencing (scRNA-seq) on mice PFC across four timepoints (postnatal day: P1, P4, P10, and adulthood: P84). Our comprehensive transcriptomic atlas captured dynamic gene expression patterns during postnatal development of neuronal maturation. By integrating these data with spatial transcriptomic profiles from juvenile and adult PFC, we systematically mapped the spatiotemporal distribution patterns of diverse neuronal subtypes. We analyzed in detail the stage-specific molecular characteristics during the maturation process of IT neurons in different layers of PFC and the dynamic expression programs of genes that regulate axon development and synaptic formation. We further revealed the developmental patterns of neuron-glia interaction network and the enrichment patterns of risk genes for neurological diseases across PFC subtypes. This comprehensive dataset provided a valuable resource for studying the development of mouse PFC, and can be viewed online through our user-friendly website: https://huggingface.co/spaces/TigerZheng/PFCdev-web . The first two postnatal weeks constitute a crucial period for IT axon growth and circuit formation: axons typically reach their target regions within the first week and undergo subsequent refinement through activity-dependent pruning [ 9 , 21 ]. Consequently, this period is essential for the proper establishment of IT-mediated dynamic neural circuits. Despite recent advances in characterizing the molecular diversity of adult PFC IT neurons [ 22 , 23 ], the transcriptional dynamics and regulatory mechanisms governing their axonal growth and synaptic integration during early postnatal period (1–2 weeks) remain poorly understood. Elucidating these mechanisms is crucial for understanding how early molecular programs shape PFC functional architecture and how their disruption may contribute to neurodevelopmental disorders such as autism and schizophrenia. Like other cortical regions, PFC follows the conserved inside-out neurogenesis pattern during embryonic development, where neurons are generated in successive waves from deep to superficial layers [ 14 , 15 ]. Corticothalamic (CT) and pyramidal tract (PT) neurons, which reside predominantly in deep cortical layers, are born early and almost complete their radial migration prenatally. These neurons are among the first to establish long-range connections with thalamic and subcortical targets [ 16 – 18 ]. In contrast, intratelencephalic (IT) neurons, the most abundant excitatory neuronal population in the cerebral cortex, distributed across both deep and superficial cortical layers, with their migration and maturation extending into the postnatal period [ 14 , 19 , 20 ]. The prefrontal cortex (PFC) serves as the central regulatory hub for higher cognitive functions in the mammalian brain [ 1 , 2 ]. Its sophisticated neural networks critically depend on the precisely coordinated spatiotemporal development of neurons [ 3 , 4 ]. Unlike other cortical regions, the PFC exhibits a prolonged maturation period that extends well into postnatal life [ 5 , 6 ]. While neuronal fate is predominantly determined by prenatal gene expression patterns, the postnatal developmental processes—including ongoing neuronal migration, axonal elongation, and synapse formation—are fundamentally essential for establishing mature neural circuits [ 7 – 9 ]. Notably, early sensory experiences, environmental changes, and stress exposure during this period can significantly influence PFC circuit development and functionality [ 10 – 12 ]. For example, maternal separation during the first postnatal week reduces the number of inhibitory neurons and synapses in mouse PFC, resulting in social deficits in adulthood [ 13 ]. Results Identification of cell-type-specific transcription factors during IT neuron postnatal development Our scRNA-seq data revealed significant transcriptomic dynamics of PFC IT neurons during postnatal development. To further investigate the heterogeneity of IT neurons during development, we reconstructed the developmental trajectory of PFC IT neurons using Monocle2 [34] (Fig 3A and 3B). Pseudotime analysis delineated the dynamic progression from immature IT neurons to mature IT neurons (Fig 3C). IT neurons at different developmental timepoints were orderly distributed along the pseudotime axis. IT neurons at P1 and P4 predominantly located at the beginning of the trajectory, while those at P10 and adult predominantly located at the end of the trajectory (S3A–S3D Fig), suggesting that IT neuron maturation is tightly synchronized with temporal progression. We observed some L6 IT neurons appear early in pseudotime. This is because deep neurons develop first, so some L6 IT neurons have already matured at P1. Through differential gene expression analysis, we identified 3,198 genes that exhibited significant alterations along pseudotime (S3 Table). These genes were clustered into five distinct modules. GO enrichment analysis revealed the function of each gene module (Fig 3D). Genes highly expressed at the beginning of the trajectory were associated with neuronal development and projection organization, whereas those enriched at the end of the trajectory were associated with cell communication and transport. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 3. The maturation dynamics and the underlying transcription factor regulation of mouse PFC IT neurons during postnatal development. (A) Uniform Manifold Approximation Projection (UMAP) of mouse PFC IT neurons at different ages during development, which is colored by pseudotime value. (B) Pseudotime trajectory of IT neurons, which is colored by time stages. (C) Pseudotime trajectory of each IT neuron subtype, which is colored by IT subtypes. (D) Heatmap shows gene modules associated with pseudotime and gene ontology terms for each gene modules. (E) Schematic illustrates the spatially specific TFs regulation of IT neurons in the immature (left) and mature (right) mouse prefrontal cortex (PFC). (F) Dotplot shows the specific TF regulons expressed in each IT neuron subtype. (G) The TF regulatory networks of relguons Sox11(+), Sox12(+), Atf4(+), Tbr1(+) and their top 20 target genes are shown respectively. Edges are colored based on importance score. The TF binding motifs are shown on the left. (H) UMAP visualization of Sox11 gene expression in IT neurons (left). Allen mouse brain ISH images of Sox11 gene at E18.5, P4, P14, and P56 (right). (I) UMAP visualization of Sox12 gene expression in IT neurons (left). Allen mouse brain ISH images of Sox12 gene at E18.5, P4, P14, and P56 (right). (J) UMAP visualization of Atf4 gene expression in IT neurons (left). Allen mouse brain ISH images of Atf4 gene at E18.5, P4, P14, and P56 (right). (K) UMAP visualization of Tbr1 gene expression in IT neurons (left). Allen mouse brain ISH images of Tbr1 gene at E18.5, P4, P14, and P56 (right). https://doi.org/10.1371/journal.pbio.3003594.g003 The development of neurons and their projection organization are under the control of transcription factors (TFs) [4]. Using SCENIC [35], we identified distinct regulons in each IT neuronal subtype (S3E Fig and S4 Table). Based on these results, we delineated a spatiotemporal TFs landscape during IT neuron maturation (Fig 3E). We found that IT neurons were regulated by distinct TFs during different mature and immature states. Immature IT neurons at P1 were primarily regulated by neurodevelopmental TFs, such as the Sox gene family, whereas mature IT neurons at adult were mainly regulated by metabolic and signaling transmission-related TFs, such as Nr1d1(+), Hes1(+), Rxrg(+). Of note, these TFs exhibit layer-specific spatial expression patterns, underlying the specific regulation of the development IT neuronal subtypes within precise locations at different developmental stages (Fig 3E and 3F). We further analyzed four TFs (Sox11(+), Sox12(+), Atf4(+), and Tbr1(+)) that regulate immature IT neurons in different cortical layers, and constructed their downstream target gene regulatory networks. These TFs bind to target genes through specific binding motifs to exert regulatory functions (Fig 3G). The spatial expression patterns of these TFs were further validated by Allen ISH data. Sox11, which has been previously reported in the regulation of circuit development of L2/3 IT neurons in the motor cortex [15], is highly expressed in immature L2/3 IT neurons at P1 (Fig 3H). Similarly, immature L4/5 IT neurons highly express Sox12 (Fig 3I), whereas Atf4 and Tbr1 are expressed in immature L5 IT and immature L6 IT neurons, respectively (Fig 3J and 3K). These findings provided new insights for understanding the transcriptional logic of IT neuron postnatal development. [END] --- [1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3003594 Published and (C) by PLOS One Content appears here under this condition or license: Creative Commons - Attribution BY 4.0. via Magical.Fish Gopher News Feeds: gopher://magical.fish/1/feeds/news/plosone/