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. 2023 Jan 6;150(1):dev201373. doi: 10.1242/dev.201373

Fig. 4.

Fig. 4.

Induced expression of constitutively active S. purpuratus MEK (Sp-caMEK.GFP) in PMCs disrupts skeletal patterning. (A) Schematic of the transactivator and responder constructs used to induce Sp-caMEK.GFP expression in PMCs. (B) Representative images of transgenic embryos expressing Sp-caMEK.GFP after overnight Dox treatment (see Fig. 2B for treatment schedule). The protein is distributed throughout the PMC syncytial network, resulting in supernumerary spicules and abnormal skeletal branching (asterisks). The number of embryos with Sp-caMEK.GFP expression that showed the abnormal skeletal branching phenotype is indicated. Top: GFP fluorescence in live embryos. Middle: GFP fluorescence overlaid onto DIC images. Bottom: polarized light images showing skeletal elements. (C) GFP and p16 immunoFISH staining of transgenic embryos expressing Sp-caMEK.GFP showed expansion of the p16 expression domain (white bars). The number of embryos expressing Sp-caMEK.GFP that showed expanded p16 expression was scored. Top: GFP-immunostained cells. Middle: Cy3-labeled p16 RNA transcripts. Bottom: fluorescence merged with Hoechst 33342 counterstain in grayscale. Scale bars: 50 μm.