Fig. 5

MagT1 and TUSC3 are required for zebrafish embryonic development. (A) Western blots of zebrafish embryo total lysates. Fertilized eggs were injected by one of two combinations of morpholinos against both MagT1 and TUSC3 (Morpholino A, Morpholino B; 100 μM each in final concentration) or buffer alone (Control). Embryos were collected after 30 h, homogenized, lysed, and anti-MagT1 and -tubulin antibodies used to recognize zebrafish proteins. Lysate from HEK cells with stable MagT1 expression (hMagT1) served as control. Sets of the same eggs were also coinjected with either 100 ng/μL of human MagT1 and TUSC3 mRNA, or 10 mM MgCl₂, as indicated. The ratio between the MagT1 and tubulin signals was calculated and normalized to the controls. (B) RT-PCR of total RNA from the embryos injected with Morpholino B, showing effects on splicing. Eggs were injected as in (A), with the Morpholino B set. (C) Embryo survival rates after single morpholino injection. 48 h after injections with 200 μM each of the morpholinos indicated, survival rates were calculated as the ratio between total hatched larvae and total eggs injected. (D) Double morpholino injections and rescue by coinjection with Mg²⁺ and human MagT1 and TUSC3 mRNA. Eggs were injected with a combination of morpholinos against MagT1 and TUSC3 as in (A and B), and survival rates were calculated as in C. For C and D, 40–70 embryos were counted for each injection. Control, mock injection with buffer. (E) Zebrafish development is arrested after morpholino treatment. Fertilized eggs were injected with a combination of morpholino A against MagT1 and TUSC3 (100 μM each in final concentration) or buffer alone. Images: 30 h stage.