Stage-specific regulation by autophagy-related transcription factors drives hematopoietic stem cell formation.

Macroautophagy/autophagy critically regulates hematopoietic stem cell (HSC) development and differentiation, yet the upstream transcriptional mechanisms governing autophagy during dynamic developmental processes remain poorly characterized. Here, we combined single-cell RNA sequencing (scRNA-seq) with metaTF to dissect six consecutive stages of murine HSC development, spanning the aorta-gonad-mesonephros (AGM) region at embryonic day (E) 10.5, the fetal liver at E12.5/E14.5, and adult bone marrow. Beyond transcript abundance alone, we found that the activity of autophagy-related transcription factors (TFs) more robustly characterized cell-type specificity, particularly distinguishing T1 and T2 pre-HSCs, and identified 32 cell-type-specific autophagy-related TFs. Stage-specific autophagy-related TF-target gene networks constructed for T1 and T2 revealed functional partitioning of the pre-HSC stage: an early T1 phase characterized by elevated autophagy activity, and a later T2 phase primarily involved in proliferation and maturation. These findings highlight the temporal regulation exerted by autophagy-related TFs during embryonic hematopoiesis and underscore the importance of autophagy in stem cell fate decision.Abbreviations: HSC: hematopoietic stem cell; TF: transcription factor.