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Blood, Vol. 110, Issue 7, 2718-2726, October 1, 2007
Functional conservation of erythropoietin signaling in zebrafish
Blood Paffett-Lugassy et al.
110: 2718
Supplemental materials for: Paffett-Lugassy et al, Vol. 110, Issue 7, 2718-2726
The microinjection of zebrafish epo mRNA into one-cell stage gata1DsRed embryos caused an increase in the number of gata1+ cells in circulation that was readily apparent by 4.5 dpf, providing strong evidence that the isolated epo clone was functionally analogous to mammalian Epo. Video 1 shows circulation of gata1+ cells in an uninjected control at 4.5 dpf. Video 2 shows increased numbers of circulating red blood cells in an embryo injected with epo mRNA at 4.5 dpf. This polycythemia continued over the next several days such that by 7 dpf, embryos exhibited increased circulation viscosity associated with cardiac regurgitation, inhibited blood flow, localized vascular stasis, and an enlarged posterior tail vein lumen. Videos S3 and S4 show uninjected controls (4 is a close view of the posterior trunk vessels) and Videos S5 and S6 show embryos injected with epo mRNA (6 is a close view of the posterior trunk vessels). As with mammals, the absence of zebrafish EpoR caused a modest decrease in primitive erythrocyte number, detected as early as 36 hpf (Figure 4B). This reduction was more apparent at 2.5 dpf, and only a few cells remained in epor morphants by 4 dpf (Figure 4B). Video 8 demonstrates the absence of circulating gata1+ cells in a 4.5 dpf gata1DsRed embryo injected with epor morpholino; an uninjected control is shown in Video 7. Imaging of gata1DsRed embryos was made with a Hamamatsu Orca-ER digital camera (Hamamatsu Photonics, Bridgewater, NJ, USA) and Openlab software (Improvision, Lexington, MA, USA).
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