Characterization of zebrafish mutant merlot as a non-mammalian vertebrate model for congenital anemia due to protein 4.1 deficiency

The zebrafish mutant merlot (mot) is characterized by onset of a severe anemia at 96 hours post fertilization. We performed whole mount RNA in situ hybridization and showed that the process of primitive erythropoiesis is not interrupted in the mot embryos. Blood analysis demonstrated that mot suffers from a severe congenital hemolytic anemia. Using the TUNEL assay, we detected apoptotic erythroid progenitors in the kidneys. We performed electron microscopic analysis and detected membrane abnormalities and a loss of the cortical membrane organization in the mot cells. We used positional cloning techniques with a candidate gene approach to demonstrate that mot encodes the erythroid specific isoform of protein 4.1R, a critical component of the red blood cell membrane skeleton. Sequence analysis of 4.1R cDNA detected nonsense point mutations in both alleles of mot resulting in premature stop codons. We performed linkage analysis and transgenic rescue experiments to provide further confirmation that the molecular defect in the protein 4.1R is the underlying cause of anemic phenotype in mot fish. This study presents the zebrafish mutant merlot as the first characterized non-mammalian vertebrate model of congenital anemia due to a defect in protein 4.1R integrity.