Investigating the TCA cycle during hematopoiesis: a temporal and spatial characterization in the Drosophila lymph gland

Abstract

Hematopoietic progenitors must balance proliferation, self-renewal, and lineage commitment, but how central carbon metabolism achieves this balance in vivo remains poorly defined. Using a comprehensive zone-specific RNAi screen targeting every step of the tricarboxylic acid (TCA) cycle in the Drosophila larval lymph gland, we reveal that progenitor maintenance requires complete TCA cycling, while growth and differentiation are governed by distinct metabolic modules. Knockdown of TCA enzymes in progenitors consistently reduced Dome⁺ progenitors and expanded plasmatocytes, indicating that cyclical TCA flux is essential for preventing premature differentiation. In contrast, loss of citrate synthase, mitochondrial Aconitase, or α-ketoglutarate dehydrogenase uniquely impaired lymph gland growth, defining a growth-specific metabolic branch. Core progenitors required TCA activity for survival but not for global differentiation control, while the intermediate and cortical zones selectively used TCA nodes to restrain terminal differentiation. Temporally controlled perturbations demonstrated that early hematopoiesis relies on two independent modules a citrate/Aconitase branch for progenitor identity and an α-KG producing α-Kdh/Gdh branch for proliferation whereas late hematopoiesis transitions to a unified cyclical TCA loop that maintains MZ to CZ homeostasis. Dietary citrate supplementation uniquely rescued CS loss during a defined early metabolic window, establishing citrate as a critical early metabolite for progenitor identity. Together, our study uncovers a previously unrecognized modular-to-cyclical transition in TCA metabolism that coordinates organismal growth, progenitor maintenance, and lineage output in vivo.