Abstract
The vertebrate brain requires precisely patterned vasculature, yet the molecular and cellular dynamics of cerebrovascular development remain incompletely understood. While single-cell studies have profiled brain vasculature, integrating these data across molecular, cellular, and architectural scales remains challenging. We combine in situ sequencing, single-cell transcriptomics, and 3D vascular reconstruction to map zebrafish brain endothelial development (3-11 dpf). Quantitative 3D analysis reveals a developmental shift from lateral vascularization to intraparenchymal angiogenesis, coinciding with blood-brain barrier (BBB) maturation. We identify 6 endothelial subtypes with conserved mammalian signatures, showing capillary endothelial cells (CapECs), the predominant intracranial subtype exhibiting stage-specific enrichment of transporter and tight junction modules. Spatial transcriptomics show progressive CapEC enrichment in mesencephalic/metencephalic regions versus arterial EC accumulation in prosencephalic vessels. Functional assays confirm BBB competency by 11 dpf, while genetic perturbation of three uncharacterized CapEC-enriched genes (slc16a1a, zgc:158423, cldc1) disrupts vascular patterning and BBB integrity. This multidimensional atlas provides an evolutionary perspective on cerebrovascular development, bridges molecular profiles with vascular architecture, and offers insights into developmental dynamics across scales.
Data availability
The data that support the findings of this study have been deposited into NCBI Gene Expression Omnibus (GEO) database with the accession number of “GSE243907” (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE243907). Source data are provided with this paper.
Code availability
We have uploaded all the research codes in the GitHub repository (https://github.com/lixy0323/cerebrovascular-atlas).
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Acknowledgements
We thank Yuezhuang Zheng and Qiumei Hong for expert technical assistance with the zebrafish facility. We also thank Chunbao Liang, Enyu Huang, and Dr. Lianyan Li for scientific discussion and technical support on this project. We thank Dr. Dong Liu for kindly sharing the Tg(slc2a1a:eGFP) zebrafish line. This work is supported by the National Natural Science Foundation of China (No. 32222028, 32470889), the National Key R&D Program of China (2024YFA1802200, 2024YFA1802600), the High-level Talents Scientific Research Start-up Funds of the Affiliated Hospital of Guangdong Medical University (2081Z20230014, 2081Z20230015), and the Special Project for Clinical and Basic Sci & Tech Innovation of Guangdong Medical University (GDMULCJC2025010, GDMULCJC2024113).
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X.L., C.W., S.K., and J.Z. designed the experiments, analyzed the data, and wrote the manuscript. M.W., X.L., J.T., and Y.Z. performed the experiments and analyzed the data. X.L. and C.W. performed the bioinformatics analysis. Z.W. and Y.R. helped with the imaging and figure preparation. C.W., D.M., and J.Z. discussed and revised the manuscript. J.Z. supervised and designed this project.
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Li, X., Ke, S., Wu, C. et al. A spatiotemporal atlas of cerebrovascular development in zebrafish. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68995-z
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DOI: https://doi.org/10.1038/s41467-026-68995-z
