Mengnan Chenga b, Liang Li Wua b, Lei Hana, Xin Huanga b, Yiwei Laia c, Jiangshan Xua b, Shuai Wanga b, Mei Lia, Huiwen Zhenga d, Weimin Feng
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Mammalian brain development is a complex process that starts from the neurulation of the early embryo to maturation and functionalization in postnatal brain development (Stiles and Jernigan, 2010). Major biological events that occur during brain development include cell proliferation, neurogenesis, cell migration, gliogenesis, and myelination, during which numerous cells are generated and migrate to specific regions to obtain corresponding functions (Jiang and Nardelli, 2016). These processes adhere to tight spatiotemporal regulation by extrinsic and intrinsic cues across different anatomical regions or layers (Martinez, 2001; Echevarria et al., 2003; Hodge and Hevner, 2011; Manuel et al., 2015). The disruption of gene expression or regulatory factors can lead to developmental dysfunction or neuropsychiatric diseases (Li et al., 2018; Parenti et al., 2020). Previous studies have mainly focused on the molecular features of specific regions; however, a complete understanding of brain development requires comprehensive characterization of cell types, molecular features, and spatial localization of each cell or gene at the whole-brain scale (Tiklova et al., 2019; Tan et al., 2021). Mouse brain development can be broadly divided into two stages, embryonic and postnatal. During the embryonic stage, starting from embryonic day 10 (E10), a large number of neurons are generated by cell proliferation and neurogenesis. Within the first week after birth, numerous glial cells are generated, which are critical for neuronal synaptogenesis. By postnatal day 7 (P7), many neurons start forming synaptic connections to build the initial neural circuits of the brain (Han et al., 2009). Besides, some brain regions undergo dramatic changes during this period. For example, neurogenesis in the dentate gyrus of the hippocampus begins at E10 and reaches a maximum rate in the first two postnatal weeks (P0–P14) (Zhang et al., 2005). Granule cells of the cerebellum begin at E15 and undergo massive proliferation, peaking from P5 to P7 (Zhang et al., 2005; Carter et al., 2018; Kozareva et al., 2021). Therefore, it is significant to understand the molecular, cellular, and regional characteristics of this stage. Single-cell and spatial transcriptomic technologies have greatly accelerated the understanding of cell type taxonomy, spatial localization, and functions in both adult and developing brains (Lein et al., 2017). For example, Zeisel et al. (2018) described the transcriptional features of 19 regions in the central nervous system and peripheral nervous system of adult mice, including the olfactory bulb, cortex, nucleus (striatum, hypothalamus, and so on), midbrain, and spinal cord. Some studies have focused on specific cortical regions of the adult brain in mice or humans (Lake et al., 2016; Tasic et al., 2016; Tasic et al., 2018). The current systematic molecular description of mouse brain development comes from the Allen Institute, which used the ISH approach (Thompson et al., 2014). Primate brain development has been systematically described which covers the stage from E40 to postnatal 48 months (Bakken et al., 2016). Whole-brain coverage of spatial transcriptome (via laser microdissection) and gene expression (via ISH) are also available for prenatal human brains (Miller et al., 2014; Ding et al., 2022). Furthermore, studies have focused on resolving the developmental features of the prenatal mouse brain, ranging from E7 to E18.5 (Di Bella et al., 2021; La Manno et al., 2021); however, a comprehensive study on the spatial organization of cell types at the maturation stage in the postnatal mouse brain is yet to be performed.The rapid evolution of cutting-edge spatial transcriptomic technologies has provided new methods to decipher complex brains (Liao et al., 2021; Ortiz et al., 2021). Although FISH-based spatial technology has allowed the study of gene expression patterns in specific regions, the limited number of detected genes and restricted brain field of view have largely hampered the global analysis of the entire brain in detail (Long et al., 2017). In this regard, spatial transcriptomic technologies based on nextgeneration sequencing, such as spatial transcriptomics (Stahl et al., 2016), Slide-seq (Rodriques et al., 2019), HDST (Vickovic et al., 2019), DBiT-seq (Liu et al., 2020), and Seq- Scope (Cho et al., 2021), have been employed in the field of brain development. However, these methods have limitations in terms of capture sensitivity, resolution, and field of view. To address these issues, we recently developed Stereo-seq, a DNA nanoball (DNB) patterned array-based technology that allows ultra-high resolution and spatial transcriptomics profiling with a large field of view (Chen et al., 2021). Stereo-seq enables the identification of subregions and cellular resolution analysis in complex tissues, and it has been applied to map spatial patterns of gene expression across whole mouse embryos with particular interests in the developing brains from E9.5 to E16.5 (Chen et al., 2021); however, no high-resolution spatial transcriptomic study has been performed on the neonatal developmental stage of the whole mouse brain. In this study, we applied Stereo-seq to generate a spatially resolved transcriptomic dataset of P7 murine brain sagittal sections near the middle line. Also, we determined the subcellular distribution of 27,330 genes across the entire section. We identified 41 anatomical regions with differentially expressed genes and enriched region-specific gene regulatory networks. Moreover, we performed image-based cell segmentation on this dataset and identified 99,365 cells, with 41 cell types localized in different regions. This study provides a comprehensive resource for future research on the cellular and molecular mechanisms of postnatal brain development. To facilitate this exploration, we created a website containing an open and interactive database (https://db.cngb.org/stomics/ datasets/STDS0000139?tab=explore).
