Elucidating how microscale molecular architecture shapes macroscale brain network organization requires integrative frameworks that link transcriptional profiles, structural connectivity, and functional dynamics across scales. However, existing evidence largely relies on cross-modal data acquired from different individuals, limiting causal and mechanistic inference. To overcome this, we performed concurrent awake optogenetic functional MRI (opto-fMRI) and spatial transcriptomics (opto-ST) within the same mice, enabling coupled analysis of neural activity and gene expression in fronto-thalamic and hippo-thalamic networks. Optogenetic stimulation of the medial prefrontal cortex (mPFC) or subiculum (SUB) evoked distinct thalamic BOLD response patterns. Multivariate spatial modeling revealed that energy metabolism and core biochemical processes underlie these GLM-derived activation patterns-a finding further supported by data-driven PCA, which identified enrichments in similar biological processes. These results suggest that conventional linear models predominantly capture linear gene-process relationships, highlighting the need for nonlinear computational approaches in fMRI analysis. Overall, our multi-omics framework reveals that genes regulating metabolic pathways underlie region-specific neurovascular coupling, thereby bridging local transcriptomics and large-scale functional imaging signatures.

Sensorineural hearing loss (SNHL) seriously affects people's lives, and the degradation of spiral ganglion neurons (SGNs) is the main cause of SNHL. Many studies have revealed that long non-coding RNAs (lncRNAs) are linked to SGNs. However, it is still unclear how lncRNAs affect damage repair in SGNs. Fluorescence in situ hybridization (FISH) and nuclear-cytoplasmic separation (NCS) assays were used to verify the cellular localization of lncRNA-HOTAIR in SGNs. Primary SGNs were dissected from the C57BL/6J male mice. We constructed sh-HOTAIR SGNs to investigate the role of HOTAIR. Target gene levels were analyzed using qRT-PCR and Western blot assays. Additionally, SGN proliferation was assessed using the CCK-8 and flow cytometry assays. Moreover, RIP and dual-luciferase assays were conducted to elucidate the interactions among HOTAIR, miRNA, and mRNA. Our findings illustrated that HOTAIR was primarily expressed in the cytoplasm, and downregulation of HOTAIR increased SGN apoptosis by approximately 30% (P < 0.001). In addition, miR-211-5p was identified as being directly downstream of HOTAIR, which could bind with miR-211-5p to modulate SGNs growt (all P < 0.05). Furthermore, miR-211-5p reduced the proliferation of SGNs (P < 0.001) and increased apoptosis (P < 0.01) by binding to the 3'-UTR of EYA3, whereas overexpression of EYA3 reversed this result (all P < 0.05). Our findings suggest that HOTAIR promotes SGN proliferation by competitively binding to miR-211-5p and regulating EYA3 expression, highlighting its potential as a novel target for sensorineural hearing loss therapies.

Unraveling the cellular and molecular characteristics of human prefrontal cortex (PFC) development is crucial for understanding human cognitive abilities and vulnerability to neurological and neuropsychiatric disorders. Here, in this study, we created a comparative repository for gene expression, chromatin accessibility and spatial transcriptomics of human and macaque postnatal PFC development at single-cell resolution. Integrative analyses outlined species-specific dynamic trajectories of different cell types, highlighting key windows and gene regulatory networks for processes such as synaptogenesis, synaptic pruning and gliogenesis. We identified regulatory correlates of the prolonged development of human PFC relative to macaques. Glial progenitors showed higher proliferation capability in humans compared to macaques, associated with distinct gene expression profiles. Furthermore, we uncovered cell types and lineages most susceptible to neurodevelopmental and neuropsychiatric disorders, focusing on transcription factors with human-specific expression features. In summary, our discoveries shed light on human-specific regulatory programs extending postnatal cortical maturation through coordinated neuronal and glial development, with implications for cognition and neurodevelopmental disorders.

Brain aging involves synapse decline, with astrocytes playing a key role in synapse homeostasis. However, the impact of astrocyte senescence on synaptic dysfunction and cognitive decline remains unclear. Here, we identified a hallmark of aging astrocytes—Senescent Astrocytic Deposits (SAD) observed at aged rodents, macaques, and human hippocampal astrocytic processes —that is associated with tripartite synapse dysfunction and memory decline. Laser capture microdissection-coupled mass spectrometry (LCM-MS), spatial transcriptome analysis and 3D electron microscopy revealed that SAD are abnormal protein deposits at the processes of ApoE-high expression astrocyte subtype and associated with dysfunctional tripartite synapses. Using a transgenic mouse ( Nrbf2 knockout) with accelerated SAD formation as a tool for genetic manipulation, we clearly demonstrated that age-dependent defect of phagocytosis at maturation stage in astrocytic drives SAD accumulation, synaptic injury and cognitive deficits. Collectively, our findings establish SAD as a mechanistic link between astrocyte senescence and synaptic damage, underscoring the critical role of astrocytic phagocytic function in preserving synaptic homeostasis and cognitive function during aging. Significance Statement This study reveals that impaired phagocytic maturation in senescent astrocytes leads to formation of SAD and impaired synaptic plasticity, identifying hippocampal astrocyte senescence as a key contributor to age-related cognitive decline. ### Competing Interest Statement The authors have declared no competing interest. Science and Technology Development Fund, Macau SAR, 0040/2024/RIB1, FDCT/005/2023/SKL, FDCT/0010/2023/AKP National Natural Science Foundation of China, No. 82271455 The University of Macau grants, MYRG-GRG2024-00238-ICMS-UMDF, MYRG-GRG2023-00089-ICMS-UMDF, SRG2023-00003-FHS, MYRG-GRG2024-00272-FHS The Ministry of Science and Technology of China, 2021ZD0200900 Yunnan Applied Basic Research Projects, 202305AH340006

With the rapid development of single-cell omics technology, its application in the field of developmental biology is becoming increasingly widespread. This article reviews the key role of single-cell omics techniques in research from model organisms to human developmental biology, revealing the importance of cell heterogeneity, dynamic monitoring of gene expression and epigenetic modifications, and constructing cross species developmental biology models. At the same time, the conservatism and diversity of single-cell omics technology in understanding human development, as well as its application in disease mechanism exploration, drug screening, and treatment strategy development, were also discussed. However, this technology still faces technical challenges such as sample preparation, data quality, and analysis complexity, as well as ethical challenges in the use and storage of human samples. This study aims to provide new perspectives and tools for the field of developmental biology, promote a deeper understanding of life processes, and provide strong support for future medical research and treatment.

The spinal cord is a crucial component of the central nervous system that facilitates sensory processing and motor performance. Despite its importance, the spatiotemporal codes underlying human spinal cord development have remained elusive. In this study, we have introduced an image-based single-cell transcription factor (TF) expression decoding spatial transcriptome method (TF-seqFISH) to investigate the spatial expression and regulation of TFs during human spinal cord development. By combining spatial transcriptomic data from TF-seqFISH and single-cell RNA-sequencing data, we uncovered the spatial distribution of neural progenitor cells characterized by combinatorial TFs along the dorsoventral axis, as well as the molecular and spatial features governing neuronal generation, migration, and differentiation along the mediolateral axis. Notably, we observed a sandwich-like organization of excitatory and inhibitory interneurons transiently appearing in the dorsal horns of the developing human spinal cord. In addition, we integrated data from 10× Visium to identify early and late waves of neurogenesis in the dorsal horn, revealing the formation of laminas in the dorsal horns. Our study also illuminated the spatial differences and molecular cues underlying motor neuron (MN) diversification, and the enrichment of Amyotrophic Lateral Sclerosis (ALS) risk genes in MNs and microglia. Interestingly, we detected disease-associated microglia (DAM)-like microglia groups in the developing human spinal cord, which are predicted to be vulnerable to ALS and engaged in the TYROBP causal network and response to unfolded proteins. These findings provide spatiotemporal transcriptomic resources on the developing human spinal cord and potential strategies for spinal cord injury repair and ALS treatment.

Doublecortin (DCX) is one of the major causal proteins leading to lissencephaly and subcortical band heterotopia in human patients. However, our understanding of this disease, as well as the function of DCX during neurogenesis, remains limited due to the absence of suitable animal models that accurately represent human phenotypes. Here, we conducted a comprehensive examination of the neocortex at different stages in DCX knockout ferrets. We corroborated the neurogenic functions of DCX in progenitors. Loss of function of DCX led to the over-proliferation of neural progenitors and the truncation of basal processes of radial glial cells, which contributed to the thickening of cortices and the stalling of neurons underneath the cortical plate during neurogenic stages, respectively. We also present the first-ever cell atlas of the lissencephaly disease model, which embraces an almost reversed neuronal lamination distribution in the neocortex compared to the normal controls. Furthermore, we discovered alterations in molecular signatures tied to epilepsy, a condition frequently observed in lissencephaly patients. We also provided compelling evidence that the distribution of GABAergic inhibitory neurons in the cortex is intricately linked to glutamatergic excitatory neurons in a subtype-specific manner. In conclusion, our research offers new insights to expand our understanding of DCX’s functions and enrich our comprehension of lissencephaly’s intricacies. Highlights DCX ferrets phenocopy human lissencephaly and subcortical band heterotopia syndrome DCX is required for NPC proliferation and radial glial basal fiber extension The atlas of lissencephalic cortex is illustrated using snRNA-seq and spatial transcriptome Inhibitory neurons couple to excitatory neurons in a cell-type specific manner ### Competing Interest Statement The authors have declared no competing interest.

The emergence of the three germ layers and the lineage-specific precursor cells orchestrating organogenesis represent fundamental milestones during early embryonic development. We analyzed the transcriptional profiles of over 400,000 cells from 14 human samples collected from post-conceptional weeks (PCW) 3 to 12 to delineate the dynamic molecular and cellular landscape of early gastrulation and nervous system development. We described the diversification of cell types, the spatial patterning of neural tube cells, and the signaling pathways likely involved in transforming epiblast cells into neuroepithelial cells and then into radial glia. We resolved 24 clusters of radial glial cells along the neural tube and outlined differentiation trajectories for the main classes of neurons. Lastly, we identified conserved and distinctive features across species by comparing early embryonic single-cell transcriptomic profiles between humans and mice. This comprehensive atlas sheds light on the molecular mechanisms underlying gastrulation and early human brain development.

The amygdala, or an amygdala-like structure, is found in the brains of all vertebrates and plays a critical role in survival and reproduction. However, the cellular architecture of the amygdala and how it has evolved remain elusive. Here, we generated single-nucleus RNA-sequencing data for more than 200,000 cells in the amygdala of humans, macaques, mice, and chickens. Abundant neuronal cell types from different amygdala subnuclei were identified in all datasets. Cross-species analysis revealed that inhibitory neurons and inhibitory neuron-enriched subnuclei of the amygdala were well-conserved in cellular composition and marker gene expression, whereas excitatory neuron-enriched subnuclei were relatively divergent. Furthermore, LAMP5(+) interneurons were much more abundant in primates, while DRD2(+) inhibitory neurons and LAMP5(+)SATB2(+) excitatory neurons were dominant in the human central amygdalar nucleus (CEA) and basolateral amygdalar complex (BLA), respectively. We also identified CEA-like neurons and their species-specific distribution patterns in chickens. This study highlights the extreme cell-type diversity in the amygdala and reveals the conservation and divergence of cell types and gene expression patterns across species that may contribute to species-specific adaptations.

Whether adult hippocampal neurogenesis (AHN) persists in adult and aged humans continues to be extensively debated. A major question is whether the markers identified in rodents are reliable enough to reveal new neurons and the neurogenic trajectory in primates. Here, to provide a better understanding of AHN in primates and to reveal more novel markers for distinct cell types, droplet-based single-nucleus RNA sequencing (snRNA-seq) is used to investigate the cellular heterogeneity and molecular characteristics of the hippocampi in macaques across the lifespan and in aged humans. All of the major cell types in the hippocampus and their expression profiles were identified. The dynamics of the neurogenic lineage was revealed and the diversity of astrocytes and microglia was delineated. In the neurogenic lineage, the regulatory continuum from adult neural stem cells (NSCs) to immature and mature granule cells was investigated. A group of primate-specific markers were identified. We validated ETNPPL as a primate-specific NSC marker and verified STMN1 and STMN2 as immature neuron markers in primates. Furthermore, we illustrate a cluster of active astrocytes and microglia exhibiting proinflammatory responses in aged samples. The interaction analysis and the comparative investigation on published datasets and ours imply that astrocytes provide signals inducing the proliferation, quiescence and inflammation of adult NSCs at different stages and that the proinflammatory status of astrocytes probably contributes to the decrease and variability of AHN in adults and elderly individuals.

Genetic variation confers susceptibility to neurodevelopmental disorders by affecting the development of specific cell types. Changes in cortical and striatal γ-aminobutyric acid–expressing (GABAergic) neurons are common in autism and schizophrenia. In this study, we used single-cell RNA sequencing to characterize the emergence of cell diversity in the human ganglionic eminences, the transitory structures of the human fetal brain where striatal and cortical GABAergic neurons are generated. We identified regional and temporal diversity among progenitor cells underlying the generation of a variety of projection neurons and interneurons. We found that these cells are specified within the human ganglionic eminences by transcriptional programs similar to those previously identified in rodents. Our findings reveal an evolutionarily conserved regulatory logic controlling the specification, migration, and differentiation of GABAergic neurons in the human telencephalon.

The human retina is a complex neural tissue that detects light and sends visual information to the brain.However,the molecular and cellular processes that underlie aging primate retina remain unclear.Here,we provide a comprehensive transcriptomic atlas based on 119 520 single cells of the foveal and peripheral retina of humans and macaques covering different ages.The molecular features of retinal cells differed between the two species,suggesting distinct regional and species specializations of the human and macaque retinae.In addition,human retinal aging occurred in a region-and cell-type-specific manner.Aging of human retina exhibited a foveal to peripheral gradient.MYO9A-rods and a horizontal cell subtype were greatly reduced in aging retina,indicating their vulnerability to aging.Moreover,we generated a dataset showing the cell-type-and region-specific gene expression associated with 55 types of human retinal disease,which provides a foundation to understanding of the molecular and cellular mechanisms underlying human retinal diseases.Such datasets are valuable to understanding of the molecular characteristics of primate retina,as well as molecular regulation of aging progression and related diseases.

During evolution, neural progenitor cells in the subventricular zone (SVZ) have fundamental functions, ranging from brain volume expansion to the generation of a six-layered neocortex. In lissencephalic animal models, such as rodents, the majority of neural progenitors in the SVZ are intermediate progenitor cells (IPCs). Most IPCs in rodents undergo neurogenic division, and only a small portion of them divide a very limited number of times to generate a few neurons. Meanwhile, in gyrencephalic animals, such as primates, IPCs are able to self-renew for up to five successive divisions. However, abundant IPCs with successive proliferative capacity have not been directly observed in nonprimate species. In this study, we examined the development of neural progenitors in the Chinese tree shrew (Tupaia belangeri chinensis), a lissencephalic animal with closer affinity than rodents to primates. We identified an expansion of the SVZ and the presence of outer radial glial (oRG) cells in the neocortex. We also found that IPCs have the capacity to self-amplify multiple times and therefore serve as major proliferative progenitors. To our knowledge, our study provides the first direct evidence of abundant IPCs with proliferative potential in a nonprimate species, further supporting the key role of IPCs in brain expansion.

Sequential generation of neurons and glial cells during development is critical for the wiring and function of the cerebral cortex. This process requires accurate coordination of neural progenitor cell (NPC) fate decisions, by NPC-autonomous mechanisms as well as by negative feedback from neurons. Here, we show that neurogenesis is protracted and gliogenesis decreased in mice with mutations of genes Celsr3 and Fzd3. This phenotype is not due to gene inactivation in progenitors, but rather in immature cortical neurons. Mutant neurons are unable to upregulate expression of Jag1 in response to cortical Wnt7, resulting in blunted activation of Notch signalling in NPC. Thus, Celsr3 and Fzd3 enable immature neurons to respond to Wnt7, upregulate Jag1 and thereby facilitate feedback signals that tune the timing of NPC fate decisions via Notch activation.

Notch signaling mediates multiple developmental decisions in Drosophila. In this study, we have examined the role of Notch signaling in Drosophila larval optic lobe development. Loss of function in Notch or its ligand Delta leads to loss of the lamina and a smaller medulla. The neuroepithelial cells in the optic lobe in Notch or Delta mutant brains do not expand but instead differentiate prematurely into medulla neuroblasts, which lead to premature neurogenesis in the medulla. Clonal analyses of loss-of-function alleles for the pathway components, including N, Dl, Su(H), and E(spl)-C, indicate that the Delta/Notch/Su(H) pathway is required for both maintaining the neuroepithelial stem cells and inhibiting medulla neuroblast formation while E(spl)-C is only required for some aspects of the inhibition of medulla neuroblast formation. Conversely, Notch pathway overactivation promotes neuroepithelial cell expansion while suppressing medulla neuroblast formation and neurogenesis; numb loss of function mimics Notch overactivation, suggesting that Numb may inhibit Notch signaling activity in the optic lobe neuroepithelial cells. Thus, our results show that Notch signaling plays a dual role in optic lobe development, by maintaining the neuroepithelial stem cells and promoting their expansion while inhibiting their differentiation into medulla neuroblasts. These roles of Notch signaling are strikingly similar to those of the JAK/STAT pathway in optic lobe development, raising the possibility that these pathways may collaborate to control neuroepithelial stem cell maintenance and expansion, and their differentiation into the progenitor cells.

During Drosophila optic lobe development, proliferation and differentiation must be tightly modulated to reach its normal size for proper functioning. The JAK/STAT pathway plays pleiotropic roles in Drosophila development and in the larval brain, has been shown to inhibit medulla neuroblast formation. In this study, we find that JAK/STAT activity is required for the maintenance and proliferation of the neuroepithelial stem cells in the optic lobe. In loss-of-function JAK/STAT mutant brains, the neuroepithelial cells lose epithelial cell characters and differentiate prematurely while ectopic activation of this pathway is sufficient to induce neuroepithelial overgrowth in the optic lobe. We further show that Notch signaling acts downstream of JAK/STAT to control the maintenance and growth of the optic lobe neuroepithelium. Thus, in addition to its role in suppression of neuroblast formation, the JAK/STAT pathway is necessary and sufficient for optic lobe neuroepithelial growth.