Fig. 5

Forced expression of prox1a does not commit venous ECs to a lymphatic phenotype. (A) Schematic of the prox1a overexpression construct and the flt4:Gal4FF line, which drives Gal4FF expression under the control of a Medaka 3.8 kb flt4 promoter fragment. (B-E) Whole-mount in situ hybridization against prox1a in uninjected flt4:Gal4FF siblings (B,C) and embryos injected with the prox1a mis-expression construct at 32 hpf (D,E). Note the domain of forced prox1a expression within the axial vessels in injected embryos (arrows in E), which does not reflect endogenous prox1a expression (C). Arrowheads in B,D highlight endogenous prox1a expression in the lateral line primordium; arrowhead in C points at a signal in the corpuscles of Stannius. (F-M) flt4:Gal4FF;UAS:eGFP wild-type siblings (F-I) and embryos injected with UAS:prox1a_IRES_mTurq-NLS-UAS:mRFP plasmid (J-M). At 2 dpf, forced expression of prox1a in ECs (marked by mRFP expression in J,K) does not lead to the emergence of ectopic secondary sprouts (F,G). At 5 dpf, UAS:prox1a-UAS:mRFP positive ECs are still evident in arterial and venous ECs without any signs of lymphatic or venous defects (L), suggesting that prox1a expression is not capable of influencing the cell fate and behavior of arterial and venous ECs (red in L,M). Arrows in F,J point at PLs. In H,L, arrows highlight the TD and ISLVs and white arrowheads in J-M mark exemplary ECs harboring the prox1a mis-expression construct. Note that the flt4:Gal4FF reporter line shows occasional expression in myotome cells as well as in a subset of neurons in the spinal cord. UIC, uninjected control.