Single-Cell Sequencing Reveals Thalidomide-Induced Teratogenicity in Zebrafish via Endoplasmic Reticulum Stress and Dysregulated Angiogenic Signaling

Thalidomide (TD) can lead to zebrafish having various malformations, including pectoral fin bud deformities, ear malformations. It induces the degradation of protein substrates, such as ∆Np63α, TAp63α, and SALL4, thereby exerting teratogenic effects on zebrafish. The degradation of these protein substrates triggers downstream changes, including oxidative stress and anti-angiogenesis. However, the genes involved in oxidative stress and anti-angiogenesis remain unclear. Here, the authors used single-cell RNA sequencing (scRNA-seq) to assess how TD exposure affects the transcriptome diversity of 69,115 cells at 1, 2, and 5 days post-fertilization (dpf). Wide-type group data sets were obtained from the NCBI SRA database (Accession: SRP221273). The authors' analysis identified 43 cell populations, with significant alterations in the gene expression profiles of 16 of these populations. Expression of the heat shock protein family gene hsp was upregulated in several cellular subpopulations (heart, vessel, pectoral fin bud, etc.), and the heat shock protein family genes were associated with stress. A protein-protein interaction (PPI) network was constructed using STRING and Cyto-scape software, and hub genes associated with TD-induced zebrafish teratogenicity were identified through the CytoHubba plugin. These genes play crucial roles in cellular stress responses, particularly the significance of heat shock proteins (HSPs) in regulating cellular stress and protecting cell survival. Functional analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed enrichment in pathways associated with endoplasmic reticulum protein processing and spliceosome. The genes her15.1, her4.3, and her4.4 exert an anti-angiogenic effect by modulating the Notch signaling pathway. The results obtained from RT-qPCR agreed with the sequencing data. This study constructed a single-cell transcriptomic atlas of zebrafish embryos under TD exposure through transcriptomic analysis at the single-cell level, supplementing the investigation of oxidative stress and anti-angiogenesis-related genes in TD-induced zebrafish malformations. Our research on TD, which causes cranio-maxillofacial-related malformations like ear and eye defects across multiple species, holds great significance for cranio-maxillofacial surgery.