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. 2019 Sep 2;28:101313. doi: 10.1016/j.redox.2019.101313

Fig. 1.

Fig. 1

Gross anatomy and microscopic observation, analysis of erythrocyte count, morphology and function of vgll4−/−zebrafish. (A) Gross anatomy of adult mutant fish showing splenomegaly and kidney paramorphia compared with wild type (wt) fish. H&E staining showing that, in spleen, lymphocytes in the blood sinuses were normal in the mutant fish (blue arrow), while the peripheral space was filled with irregular erythrocytes, which were almost completely absent in wildtype animals (red arrow). Original magnification, × 600. Scale bar, 25 μm. In the mutant pronephros, the erythroid series was significantly advanced in control wild type samples (red arrow). Original magnification, × 400. Scale bar, 25 μm. Erythrocyte-counts were significantly increased in both the spleen and renal marrow of vgll4b-/- zebrafish. (B) Erythrocyte count comparisons between wt and vgll4b mutants in peripheral blood from 6-month-old adult zebrafish or single cell suspensions of 3 dpf embryos. Comparison of heme levels between wt and vgll4b mutants in peripheral blood samples from 6-month-old adult zebrafish or single cell suspensions of 3 dpf embryos (left and right, respectively). (C) Wright–Giemsa stain of 3 dpf embryos demonstrating slightly more immature erythrocytes in vgll4b mutants, with round, light colored nuclei. Wright–Giemsa stain of 5 dpf embryos, showing the pale cytoplasm and immature cell morphology compared with wild-type embryos. Methylene blue staining of mutant erythrocytes revealing that the cytoplasm contained hyperchromatic dots, resembling cytoplasmic DNA, while the nuclei were less compact, indicating an immature morphology compared with control cells. (D) Prussian Blue staining of the spleen showing few iron deposits in the tissue in vgll4b-/- zebrafish. (E) Transmission electron microscope image of erythrocytes isolated from control and vgll4b-/- zebrafish at 3 dpf. Erythrocyte was fusiform and Erythroid mitochondria were observed with normal membranes in WT, while in vgll4b mutants, erythrocyte was rounder shaped and immature, with swollen ruptured-membrane mitochondria. Erythrocyte-counts were calculated from three fields of view per fish, and six fish per group. All features are indicated by red arrows. Scale bar is 25 μm in optical micrograph and 1 μm in TEM micrograph. The statistical significance of differences between data points (mean ± s.e.m.) is denoted by *P < 0.05, **P < 0.01, and ***P<0.001; NS, no significant difference. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)