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

Fig. 5.

Fig. 5.

Induced expression of S. purpuratus DUSP6 (Sp-DUSP6.GFP) in PMCs inhibits skeletogenesis. (A) Schematic of the transactivator and responder constructs used to induce Sp-DUSP6.GFP expression in PMCs. (B) Representative images of transgenic embryos expressing Sp-DUSP6.GFP after overnight Dox treatment (see Fig. 2B for treatment schedule). The protein is distributed throughout the PMC syncytial network, inhibiting spicule formation and skeletal growth (asterisks). The number of embryos with Sp-DUSP.GFP expression that showed an abnormal skeletal growth and elongation phenotype was scored. 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-DUSP6-GFP in PMCs showing loss of p16 expression (asterisks). The number of embryos expressing Sp-DUSP6.GFP that exhibited reduced p16 expression was scored. Top: GFP-immunostained cells. Middle: Cy3-labeled p16 RNA transcripts. Bottom: fluorescence merged with Hoechst 33342 counterstain in grayscale. (D) GFP and sm30b immunoFISH staining of transgenic embryos expressing Sp-DUSP6.GFP showing partial loss of sm30b expression in some PMCs (white bars). The number of embryos expressing Sp-DUSP6.GFP that exhibited reduced sm30b expression was scored. Top: GFP-immunostained cells. Middle: Cy3-labeled sm30b RNA transcripts. Bottom: fluorescence merged with Hoechst 33342 counterstain in grayscale. Scale bars: 50 μm.