rasa3 knockdown in zebrafish recapitulates the scat anemia and thrombocytopenia phenotype. (A) In WT embryos, abundant ¿-dianisidine positive hemoglobinized cells are seen in the heart and vessels (arrow) at 72 h postfertilization, whereas positive cells are absent in rasa3 morphants (MO). (B) Quantitation of the range of embryos containing normal and reduced or null number of hemoglobinized cells at 72 h postfertilization from control (uninjected) and rasa3 MO (injected). X ± SEM; *P < 0.05, t test. (C) By RT-PCR, abnormally processed rasa3 mRNA splice forms are found in MO compared with RNA control (CTRL) and uninjected embryos (WT). actb serves as a control of off-target effects. (D) Silencing of rasa3 results in loss of GFP+ cd41-thrombocytes (green) in the transgenic Tg(cd41:GFP) line (MO) compared with control (WT). (E) Quantitation of GFP+ cd41-thrombocytes by flow cytometry in control and rasa3 morphant (MO) embryos. X ± SEM; *P < 0.05, t test. (F) Treatment of WT zebrafish (uninjected) and rasa3 MO (injected) from the Tg(globin LCR:eGFP) transgenic line with the potent antioxidant N-acetyl cysteine (+NAC) fails to improve anemia in the rasa3 MO embryos, indicating that generation of ROS per se is not a contributing factor in the anemia. NAC, exposed to vehicle carrier only. X ± SEM; *P < 0.05; **P < 0.01, t test.
Anemic phenotype of rasa3 morphants and expression of rasa3 in developing zebrafish. (A) rasa3 is widely expressed in the developing zebrafish embryo (Left), in contrast to the tissue-restricted expression of gata-1 to the blood island (intermediate cell mass, Right, arrow). (B) Defect in hemoglobinization is revealed in rasa3 morphants (Right) with o-dianisidine staining at 48 hpf.
|Acknowledgments:||ZFIN wishes to thank the journal Proceedings of the National Academy of Sciences of the United States of America for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Proc. Natl. Acad. Sci. USA|