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Characterization of zebrafish merlot/chablis as non-mammalian vertebrate models for severe congenital anemia due to protein 4.1 deficiency

¹ Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912. USA
² Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA
³ Howard Hughes Medical Institute, Children’s Hospital of Boston, 320 Longwood Avenue, Enders 750, Boston, MA 02115, USA

View larger version (52K): [in a new window]   Fig. 1. Characterization of the embryonic phenotype in mot/cha. (A) Whole-mount o-dianisidine staining of wild-type and mot/cha embryos; (top) at 48 hpf, wild-type and mot embryos have a similar number of blood cells; (middle) at 96 hpf, mot embryos lack circulating blood cells (arrow); (bottom) wild-type and cha embryos at 72 hpf are stained with o-dianisidine for hemoglobin in the cardiac sinus (arrows). (B) After the onset of anemia, excretion of bile pigments in mot embryos is noticeable (arrow) and continues for several days. (C) Wright-Giemsa staining of circulating red cells collected at 48 hpf from wild-type and mot embryos reveals the presence of cells with abnormal morphology in the mot embryos. Wild-type cells are spherical with round, open nuclei, but mot contains binucleated cells (arrow), and morphologically abnormal cells with condensed nuclei and spiculated membranes. (D) Whole-mount RNA in situ analysis of wild-type and mot embryos with the hematopoietic genes, scl, gata1 and globin reveals comparable levels of transcript expression in wild-type and mot embryos at 24 hpf, indicating that erythropoiesis in mot fish is not interrupted. Lateral views are illustrated with anterior towards the left and dorsal towards the top.

View larger version (68K): [in a new window]   Fig. 2. Hematological analysis and gross anatomy of adult mot. (A) Wild-type red blood cells are terminally differentiated with elliptical morphology, condensed nuclei and hemoglobin-filled cytoplasm. Blood cells collected from adult mot fish exhibit a maturation arrest at the late basophilic erythroblast stage and striking membrane abnormalities. (B) Wright-Giemsa staining of a tissue preparation of the wild-type and mutant fish kidneys reveals an erythroid hyperproliferation in the mot fish with a drastically decreased myeloid/erythroid ratio. A few cells at the early proerythroblast stage also show membrane spiculation. (C) Peripheral blood cells from wild-type zebrafish are compared with concentrated peripheral blood cells from adult cha fish, which show abnormal morphology and differentiation arrest. (D) Gross anatomy of mot fish shows a dilated cardiac chamber compared with wild type. An icteric liver, greatly enlarged kidney and splenomegaly are always present in the adult mot fish. (E) Mean cell volume of red blood cells of wild-type and mutant fish, as measured by an automated Coulter Gen S instrument, shows that wild-type cells have a uniformly distributed volume, whereas mot cells show a significant variation in size. The anisocytosis in mot cells is due to random membrane fragmentation of mot cells.

View larger version (15K): [in a new window]   Fig. 3. Osmotic fragility of wild-type and mot red blood cells. Red cells of five wild-type and three mutant fish were subjected to the osmotic fragility test (results represent means±s.e.m.). Wild-type cells exhibit a sigmoid curve when exposed to hypotonic solutions. The red blood cells from mot fish are extremely sensitive to osmotic stress and show an increased fragility.

View larger version (75K): [in a new window]   Fig. 4. Apoptosis and analysis of red cell membrane and marginal band. (A) A fluorescent confocal TUNEL assay on peripheral blood and kidney from wild-type and mot cells reveals a significant number of apoptotic cells (green) in the kidneys of mot, whereas peripheral blood of mutant fish showed a few cells undergoing apoptosis. (B) Scanning electron micrographs show wild-type cells with biconcave elliptical morphology. Red cells from mot fish appear microspherocytic, with abnormal membrane pitting and projections. (C,D) Transmission EM analysis reveals elliptical wild-type cells with elongated nuclei and compact, organized cortical membranes. The spherical mot cells with spiculated membranes lack a sharply packed and organized cortical membrane. (E) A confocal immunofluorescence analysis detects the localization of microtubules in a marginal band in wild-type red cells (green), whereas in mutant cells the microtubules exhibit a diffuse localization. Scale bar: in B, 2.5 µm.

View larger version (33K): [in a new window]   Fig. 5. Identification of protein 4.1R as the mutated gene in mot/cha. (A) cDNA sequence comparison of wild-type and both alleles of mot shows two nonsense point mutations in the mot alleles (point mutations are underlined). The DNA sequence of mottm303c and its corresponding wild-type sequence shown here are in the 3'to 5' orientation. (B) A protein truncation test shows the truncated translation product of mottu275 cDNA compared with that of wild-type cDNA. (C) Linkage analysis via allele specific PCR primers on genomic DNA from wild-type (lanes 1-4), heterozygous (lanes 5-8) and mot (lanes 9-12). The wild-type primer, top, amplifies wild-type and heterozygous DNA, whereas the mutant primer, bottom, amplifies DNA from heterozygous and mot embryos. (D) Genetic map of the cha locus on LG16. EST Fb70c02 showed no genetic recombinants from the cha locus in 127 informative animals. (E) Allele-specific oligonucleotide hybridization for wild-type and mutant protein 4.1R sequences. Wild-type siblings (lanes 1-7) and mutant siblings (lanes 8-14) show complete linkage with either normal or mutant protein 4.1R alleles, respectively. (F) P4.1R in situ hybridization shows an expression pattern restricted to the hematopoietic intermediate cell mass. Wild-type 24 hpf embryos exhibit high-level expression of the P4.1R transcript, whereas the level of transcript expression is greatly reduced in mot embryos. (G) At 96 hpf, mot embryos that were injected with P4.1R/GFP construct are partially rescued and have a higher number of circulating red cells compared with mot controls.

View larger version (106K): [in a new window]   Fig. 6. Amino acid sequence of zebrafish protein 4.1. The BLAST application was used to perform a pair-wise amino acid alignment of protein 4.1 in human and zebrafish, and to identify putative functional domains of zebrafish P4.1. Amino acids 1-330 (yellow) correspond to the N-terminal FERM domain; 400-1400 correspond to the 16 kDa domain; 1400-1513 (red) correspond to the spectrin binding domain; and the last 21 amino acids correspond to the 20 kDa domain of human protein 4.1. The four tandem repeats starting at amino acid 780 and ending at 1106 are indicated in green and blue.

View larger version (91K): [in a new window]   Fig. 7. Amino acid alignment and phylogenetic tree of P4.1R family members. (A) Alignment of the conserved FERM domain in P4.1R proteins from zebrafish, human, mouse, Xenopus and Drosophila (Dm coracle). Shaded blocks indicate similar and identical amino acids. (B) A phylogenetic tree of the protein P4.1R amino acid sequences reveals an evolutionary conservation between members of P4.1R superfamily. The MegAlign application in DNAStar software was used for alignment and construction of the phylogenetic tree.