Pou3f2 knockdown in the tg(fli1:EGFP)^y1 zebrafish embryo.

(a) Bright-field images of embryos injected with caged morpholino against Pou3f2 translation start site in the absence of photoactivation (control), or with photoactivation with UV light at 6 or 24 hpf. (b) Fluorescence images of embryos at 48 hpf. Experimental groups were injected with caged morpholino against Pou3f2 in the absence of photoactivation (control), or with photoactivation with UV light at 6 or 24 hpf, or with photoactivation at 6 hr in the presence of rescue mRNA encoding Pou3f2. (c) Quantitation of the number of intersegmental vessels in 20 somites in embryos at 48 hpf. (d) In situ hybridization with antisense RNA probes specific for Kdr and Fli1 in whole zebrafish embryos 28 hpf. (e) Western blotting showing the reduction level of Pou3f2 following morpholino injection and rescue by mRNA encoding Pou3f2. β-Tubulin was used as loading control. ISV – Intersegmental Vessels; hpf – hour post fertilization. (f and g). Representative FACS plot and scatter plot showing a significant reduction of GFP⁺ cells in Pou3f2 KD embryos. GFP⁺ cells were sorted following isolation by enzymatic digestion from tg(fli1:EGFP)^y1 zebrafish embryos at 24 hpf. All data represented as mean ± S.E.M. N = 3. Student t-test, *p=0.01; ***p=0.001.

In-situ hybridization for Pou3f2 in zebrafish embryos.

Sense and Pou3f2-specific antisense RNA probe shows high expression of Pou3f2 in the head region, including the eye, hindbrain, midbrain and forebrain. Sense RNA probe was used as negative control.