DMT1 regulates systemic trace metal handling and developmental outcomes: Insights from targeted mutagenesis in...

Divalent metal transporter 1 (DMT1) is thought to be the primary route for non-heme iron absorption in vertebrates, but its systemic role remains poorly understood. Using CRISPR-Cas9 gene editing, we generated a DMT1 knockout (dmt1^-/-; KO) zebrafish mutant line to examine the developmental and physiological consequences of DMT1 loss. Phenotypic and hematological assessments were performed alongside measurements of whole-body and tissue-specific metal concentrations. Further, to identify potential compensatory pathways during DMT1 loss, the expression profile of candidate metal transporters or ion channels (hcp1, zip4, zip8, zip14, and ecac) was quantified using droplet digital PCR (ddPCR). DMT1 KO larvae exhibited delayed development, anemia, and broad disruption in multiple trace metals (iron, zinc, manganese, cobalt, and selenium). Gene expression analysis during early development revealed higher hcp1 mRNA abundance in the mutant, suggesting a possible compensatory response to maintain metal homeostasis during DMT1 loss. Although viable to adulthood, the mutants had persisting iron dysregulation and red blood cell abnormalities. This study provides the first in vivo evidence of the physiological role of DMT1 in multi-metal balance in fish and offers new insight into compensatory mechanisms underlying DMT1 deficiency.