Single-cell transcriptomics identifies neural fate disruption and glial reprogramming caused by <i>RARS2</i> deficiency.

Wei, Xing; Wang, Jing; Wang, Yanyun; Zhou, Cong; Chen, Zhaoyi; Liu, Shanling; Sun, Huaqin; Zhou, Bin et al. · J Med Genet · 2026

basic_science · Level V

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Abstract

Pontocerebellar hypoplasia type 6 (PCH6) is caused by biallelic pathogenic variants in <i>RARS2</i>, encoding mitochondrial arginyl-tRNA synthetase. Although mitochondrial dysfunction is a recognised feature, how <i>RARS2</i> deficiency disrupts neural lineage development remains unclear. We generated <i>rars2</i>-deficient zebrafish using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system and performed single-cell RNA sequencing (scRNA-seq) at 48 hours postfertilisation, complemented by immunofluorescence, in situ hybridisation, behavioural assays and ultrastructural analyses. Neural lineage composition, developmental trajectories, intercellular communication and transcriptional programmes were systematically examined. <i>rars2</i> <sup><i>-/-</i></sup> zebrafish displayed impaired survival, locomotor deficits, early mitochondrial ultrastructural damage and marked disruption of neurogenesis. scRNA-seq revealed reduced neuronal populations and expansion of neural progenitor and glial-like cells. Key neurogenic regulators (<i>neurod4</i>, <i>her6</i> and <i>pou3f1</i>) were downregulated, whereas glial and stress-associated markers (<i>hmgb1a, fabp7a</i> and <i>foxp1b</i>) were upregulated. Developmental pathways including Notch and non-canonical Wnt were attenuated while extracellular matrix (ECM), adhesion and inflammatory programmes were activated. Additional trajectory-based analyses supported dysregulated lineage progression characterised by glial programme activation and impaired maintenance of neurogenic differentiation. <i>RARS2</i> deficiency disrupts mitochondrial integrity and reprograms neural lineage development through coordinated suppression of neurogenic transcriptional networks and activation of glial/ECM programmes. These findings provide mechanistic insight into loss-of-function <i>RARS2</i> deficiency and highlight candidate molecular pathways for future therapeutic investigation.