Hopx is a dual-gatekeeper of astrocyte fate enabling in vivo reprogramming and neural repair

Astrocyte-to-neuron conversion provides a potential strategy for neural repair, but the endogenous mechanisms that maintain astrocytic identity and restrict neuronal fate acquisition remain incompletely understood. In this study, we investigated the role of Hopx, an astrocyte-enriched transcriptional regulator, in maintaining astrocyte fate and limiting astrocyte-to-neuron conversion. We developed an AAV-compatible TIGR interference (TIGRi) system to repress Hopx expression in vivo and applied this strategy in Aldh1l1CreERT2;Rosa26-LSL-tdTomato lineage-tracing mice. To define the transcriptional changes associated with Hopx knockdown-induced astrocyte-to-neuron conversion, we performed single-cell RNA sequencing on hippocampal cells collected after AAV-TIGRi-Hopx delivery and tamoxifen induction. scRNA-seq dataset was generated to characterize the transcriptomic states of virus-transduced astrocyte-lineage cells, identify intermediate cell populations during conversion, and reconstruct the trajectory from astrocyte-like cells toward progenitor-like and neuron-like states. The analysis revealed Hopx knockdown-associated induction of neurogenic and neuronal transcriptional programs in AAV-transduced astrocyte-lineage cells, including increased expression of progenitor- and neuron-associated markers and gene modules related to generation of neurons and neuron fate commitment. These data support the conclusion that Hopx repression promotes a progressive transcriptional transition from a glial state toward a neuron-like state and provide a transcriptomic resource for studying endogenous regulation of astrocyte plasticity during in vivo neural reprogramming. In parallel, we performed HOPX ChIP-seq to define the genome-wide binding landscape of HOPX and identify HOPX peak-associated candidate genes. Together, the scRNA-seq and ChIP-seq datasets provide complementary resources for investigating the transcriptional and genomic regulatory mechanisms underlying astrocyte-to-neuron conversion in vivo.