演化发育生物学（Evolutionary developmental biology, Evo-devo）聚焦演化和发育的互作，即演化压力如何影响物种的发育机制以及发育过程如何驱动物种的演化路径。大脑作为动物体最为复杂的器官之一，在细胞组成及基因调控上存在高度的复杂性与动态性。大脑的演化与发育研究有助于阐明中枢神经系统在稳态和疾病发生中的基因调控网络和关键信号通路。解析物种间大脑发育调控的保守性与特异性对理解神经系统起源与演化、人类认知功能及神经疾病至关重要。单细胞测序为精准识别大脑细胞亚型并追踪神经系统发育轨迹提供了有力工具。目前针对神经系统的单细胞研究主要集中在人类和模式动物（猕猴、小鼠、斑马鱼等）。然而，在大脑的演化发育研究中存在以下三大问题有待解决：①如何构建非模式物种精细的大脑发育图谱？②如何有效整合异质性的跨物种数据并进行标准化比较分析？③如何开发一个便捷、标准化的生物信息学在线平台，以促进跨物种细胞图谱的比较分析。本论文旨在构建非模式生物（鸡，Gallus gallus）的大脑发育图谱，进而整合多物种大脑图谱，并开发跨物种细胞图谱在线分析平台，系统研究脊椎动物大脑发育的调控机制。

作为脊椎动物重要的演化分支，鸟类大脑发育的细胞谱系和分子特征尚未在单细胞分辨率得到完整解析。本论文选取鸡（Gallus gallus）作为研究对象，采集了鸡胚在受精后第7至18天（E7、E10、E11、E12、E14、E16、E18）共7个关键时间点的全脑组织样本并进行单核转录组测序。通过整合公共数据与自产数据，构建了覆盖鸡大脑主要发育阶段（E6-E19, Adult）的单细胞图谱，对放射状胶质细胞、兴奋性神经元前体细胞和兴奋性神经元等关键细胞群体的功能特征与分化轨迹进行了探究。

通过结合鸡大脑发育数据与来自公共平台的人（Homo sapiens）、小鼠（Mus musculus）和斑马鱼（Danio rerio）的大脑发育单细胞图谱，构建了涵盖四个代表性脊椎动物物种的整合发育图谱。通过比较分析，评估了大脑主要细胞谱系在不同物种间的保守性与特异性，尤其关注了细胞类型的组成比例和发育动态的物种间差异。分析结果提示，人类大脑发育过程中神经元中间祖细胞（Neuron intermediate progenitor cells, nIPCs）的细胞比例与数目远高于其他三个物种。相关性分析显示8个nIPC_human在热图上明显区分于与其他nIPC亚群，nIPC_human亚群显著富集于调控转录、翻译和细胞周期的通路，以及与能量代谢和细胞稳态相关的通路，这可能与其增强的增殖能力以及干性维持有关，与皮层发育、高级认知等功能相关的通路也表现出显著富集。通过对神经元的再聚类还发现兴奋性神经元与抑制性神经元在演化过程中的差异保守性。

在此基础上，本论文系统性地整理了人类、家猪、小鼠、鸡、斑马鱼、非洲爪蟾、文昌鱼、海鞘、线虫、太平洋紫海胆、果蝇11个物种的公共单细胞/单核转录组数据，重点关注演化、发育及相关疾病研究领域的数据集，构建了SPEED在线数据库。提供针对18个演化数据集、29个发育数据集和85个疾病数据集的单细胞图谱的综合分析。

综上所述，本论文通过生成关键演化节点物种的大脑发育单细胞图谱、开展跨物种整合比较分析、构建跨物种单细胞图谱在线分析平台，为系统理解物种大脑发育的调控机制提供了数据基础和比较框架，为深入探讨神经系统演化发育的细胞异质性与分子调控机制提供了线索。

Evolutionary developmental biology (Evo-devo) investigates the interplay between evolution and development, specifically how evolutionary pressures shape the developmental mechanisms of species and how developmental processes, in turn, drive evolutionary trajectories. The brain, one of the most complex organs in animals, is characterized by remarkable complexity and dynamism in its cellular composition and gene regulatory networks. Investigating the evolution and development of the brain is crucial for understanding the gene regulatory networks and key signaling pathways governing the central nervous system in both homeostatic and disease states. Deciphering the conserved and species-specific regulatory mechanisms underlying brain development across species is essential for understanding the origin and evolution of the nervous system, human cognitive functions, and neurological diseases. Single-cell sequencing technologies enable the precise identification of brain cell subtypes and the tracking of neurodevelopmental processes. However, current single-cell studies of the nervous system have predominantly focused on humans and established model organisms (e.g., macaque, mouse, zebrafish). Significant challenges remain in brain evo-devo research: (1) constructing comprehensive developmental atlases for non-model organisms; (2) effectively integrating heterogeneous cross-species datasets for standardized comparative analysis; and (3) developing user-friendly, standardized online bioinformatics platforms to facilitate comparative analyses of cross-species cell atlases. This study aimed to address these gaps by constructing a developmental brain atlas for a non-model organism, the chicken (Gallus gallus); integrating multi-species brain atlases; and developing an online platform for cross-species atlas analysis, in order to systematically investigate the regulatory mechanisms underlying vertebrate brain development.

Birds represent a key evolutionary lineage of vertebrates, yet the cellular lineages and molecular features of avian brain development have not been comprehensively characterized at single-cell resolution. In this study, we focused on chicken (Gallus gallus) brain development. Briefly, we collected whole-brain tissue samples from chicken embryos at seven key developmental time points between embryonic day 7 (E7) and E18 (specifically, E7, E10, E11, E12, E14, E16, E18) and performed single-nucleus RNA sequencing. By integrating publicly available datasets with our newly generated data, we constructed a comprehensive single-cell atlas spanning major developmental stages of the chicken brain (E6-E19, adult). We investigated the functional characteristics and differentiation trajectories of key cell populations, including radial glia, excitatory neuron precursor cells, and excitatory neurons.

We integrated our chicken brain developmental data with publicly available single-cell atlases of brain development from human (Homo sapiens), mouse (Mus musculus), and zebrafish (Danio rerio) to build an integrated developmental atlas encompassing these four representative vertebrate species. Through comparative analyses, we evaluated the conservation and specificity of major brain cell lineages across these species, focusing particularly on inter-species differences in cell type proportions and developmental dynamics. Our analyses revealed that the proportion and abundance of neuronal intermediate progenitor cells (nIPCs) during human brain development are significantly higher compared to the other three species. Further analysis identified distinct human-specific nIPC subclusters (nIPC_human) that clustered separately from other nIPC populations. These subclusters exhibited significant enrichment for pathways regulating transcription, translation, and the cell cycle, as well as those involved in energy metabolism and cellular homeostasis, potentially reflecting enhanced proliferative capacity and stemness maintenance. Notably, pathways associated with cortical development and higher cognitive functions were also significantly enriched in these human-specific nIPC populations. Re-clustering of neuronal populations also revealed differing degrees of evolutionary conservation between excitatory and inhibitory neurons.

Building upon these findings, we systematically curated and integrated publicly available single-cell/single-nucleus transcriptomic data from 11 species (including human, pig, mouse, chicken, zebrafish, african clawed frog, amphioxus, sea squirt, elegans, pacific purple sea urchin, and fruit fly), with a focus on datasets relevant to evolution, development, and associated diseases. This effort resulted in the construction of the SPEED (Single-cell Pan-species Exploration of Evolution, Development, and Disease) online database. SPEED hosts and facilitates the integrated analysis of curated single-cell atlases from 18 evolution-focused, 29 development-focused, and 85 disease-relevant datasets.

In conclusion, by generating a single-cell brain development atlas for a key phylogenetically positioned species (chicken), performing cross-species integrative comparative analysis, and developing an online platform for cross-species single-cell atlas exploration (SPEED), this research establishes a foundational dataset and comparative framework for the systematic understanding of regulatory mechanisms underlying brain development across species. It further provides valuable resources and insights for in-depth investigations into the cellular heterogeneity and molecular regulatory principles governing nervous system evolution and development.