Evaluation of the Role of Iron on Physiological Performance and Functional Characterization of Major Iron Transporter in Zebrafish (Danio Rerio)

Abstract

Iron is essential for a multitude of biological processes, and its acquisition and regulation are critical for survival. In fishes, iron is obtained from the water via the gills and the diet through the gastrointestinal tract. While the diet is the predominant source of iron absorption, the physiological consequences of dietary iron availability and its underlying mechanisms of uptake and metabolism remain poorly understood compared to waterborne studies. In this thesis, I investigated the effects of dietary iron levels (~11, 420, and 2300 mg Fe/kg) and exposure duration (20 and 40 days) in zebrafish (Danio rerio). Short-term iron supplementation enhanced aerobic scope, maximum metabolic rate, and critical swimming speed, as well as reproductive output and offspring survival. In contrast, prolonged high-iron exposure led to iron loading in the brain and intestine, highlighting a narrow window between nutritional benefits and overload. Offspring of high-iron-fed parents exhibited greater swimming and metabolic performance than those of the low-iron group, revealing potential intergenerational consequences of parental iron status. To help elucidate the molecular mechanisms underlying iron homeostasis, a CRISPR-Cas9 knockout of the iron transporter, divalent metal transporter 1 (DMT1; slc11a2), was generated. The systemic consequences of DMT1 loss in developing and adult zebrafish were characterized using trace metal analysis, gene expression, and RNA sequencing. During early development, DMT1 loss caused anemia and disrupted iron, zinc, cobalt, and manganese homeostasis. Despite these disruptions, mutants displayed remarkable physiological plasticity and partial recovery through compensatory regulation of alternate transporters, including heme carrier protein 1 (hcp1). Broad transcriptional reprogramming was also evident in the gill and intestine of adult dmt1-/- fish, which included induction of iron transport, storage, and erythropoietic genes, alongside suppression of hepcidin/BMP-Smad signalling. Tissue-specific responses identified two candidates for DMT1-compensation, including zrt- and irt-like protein 4 (zip4) in the intestine and epithelial calcium channel (ecac) in the gills. Broad reprogramming of ion regulation, immunity, and redox pathways was also present. Collectively, this thesis advances our understanding of iron metabolism from cellular transport to whole-animal physiology, providing a framework with implications for environmental toxicology, aquaculture nutrition, and a vertebrate model for studying iron-related disorders.