Spatial transcriptomic and single-nucleus analysis reveals heterogeneity in a gigantic single-celled syncytium

Area of Science:

• Cellular and Molecular Biology
• Developmental Biology
• Eukaryotic Microbiology

Background:

• Multicellular organisms rely on cell specialization for complex body plans.
• Eukaryotic protists like slime molds form complex structures in a multinucleate syncytial state.
• The transcriptional plasticity and nuclear heterogeneity within syncytial protists remain largely unexplored.

Purpose of the Study:

• To investigate regional transcriptional differences in the slime mold *Physarum polycephalum* plasmodium under varying environmental conditions.
• To analyze gene expression heterogeneity among nuclei within the syncytium using single-nucleus RNA-sequencing.
• To compare nuclear heterogeneity in syncytial slime molds with gene expression states in single-nucleus amoebal cells.

Main Methods:

• Spatial transcriptome analysis of *Physarum polycephalum* plasmodium.
• Single-nucleus RNA-sequencing (snRNA-seq) of nuclei within the plasmodium.
• Analysis of gene expression states in single *Physarum* amoebal cells during mitosis and meiosis.

Main Results:

• Transcriptome regionality was identified in the slime mold, correlating with proliferation, syncytial substructures, and local environmental cues.
• Nuclei within the plasmodium exhibit transcriptional heterogeneity, suggesting roles in coordinating local cell growth, metabolism, and reproduction.
• Single *Physarum* amoebal cells displayed cell cycle-specific states, but lacked the predefined transcriptomic states observed in the syncytium.

Conclusions:

• The single-celled slime mold *Physarum polycephalum* exhibits region-specific gene expression control despite lacking cellular compartmentalization.
• Nuclei within the syncytium appear to function as mobile processors, facilitating localized specialized functions.
• Slime molds provide a model system to study the evolution of regulatory mechanisms for division of labor and specialization in organisms.