Characterization and functional analysis of AgRunx in the blood clam (Anadara granosa): implications for hemocyte proliferation

The Runx family of transcription factors, evolutionarily conserved regulators of vertebrate hematopoiesis, coordinates hematopoietic lineage commitment through DNA-binding Runt domains. While their roles are well characterized in vertebrates, functional insights into bivalve hemocyte proliferation remain limited. Here, we identified a Runx homolog (AgRunx) in the blood clam Anadara granosa, encoding 556 amino acid protein with a conserved Runt domain. Spatial expression analysis revealed predominant AgRunx transcription in hemolymph (10.12-fold versus gonads, P<0.05), with dynamic upregulation peaking during the D-shaped veliger developmental stage. Furthermore, AgRunx was significantly upregulated at both the mRNA and protein levels in the hemolymph of A. granosa with high total hemocyte count group [HTHC: 32.70(±2.36)×104 cells μl-1], compared with individuals with low hemocyte count group [LTHC: 7.90(±0.85)×104 cells μl-1]. AgRunx RNA interference (RNAi) showed both the total hemocyte count and hemoglobin concentration were significantly reduced. Moreover, a decrease in the number of EdU-labeled proliferating cells in gill tissues was observed, accompanied by downregulation of the G0/G1 phase and upregulation of the G2/M phase in hemocytes. These results conclusively demonstrate AgRunx is involved in the hematopoietic process of A. granosa and plays a critical role in cell proliferation.