Fig. 7.

Dual-input Tet-On system for nitroreductase-mediated cell ablation in sea urchin embryos. (A) Schematic of the transactivator and responder constructs used to induce nitroreductase (NTR-2.0.GFP) expression in PMCs. (B) Experimental design showing the treatment schedule (a,b). Solid circles indicate the stage at which Dox was added. Diamond represents the stage at which metronidazole (MTZ) was added. Arrowhead indicates the stage at which embryos were collected for analysis after overnight Dox treatment. (C) Induced expression of NTR-2.0.GFP and the addition of MTZ resulted in targeted ablation of PMCs and inhibition of skeletogenesis (asterisks). Top: GFP fluorescence in live embryos. Middle: GFP fluorescence overlaid onto DIC images. Bottom: polarized light images showing skeletal elements. The number of embryos with phenotypes similar to the representative images was scored. (D) PMC marker (MSP130) and GFP IF staining of transgenic embryos expressing NTR-2.0.GFP in PMCs. Expression of NTR-2.0.GFP and in combination with MTZ treatment disrupted the PMC syncytial cables. The number of embryos with phenotypes similar to the representative images was scored. Top: GFP-immunostained cells. Middle: MSP130-immunostained skeletal structures (6a9 antibody). Bottom: fluorescence merged with Hoechst 33342 counterstain in grayscale. Scale bars: 50 μm.