Figure 2
Figure 2. Mmp2 knockdown impairs TD formation in zebrafish. (A) Mmp2 mRNA expression (purple staining) analyzed by whole-mount in situ hybrization in 3 days postfertilization (dpf) zebrafish embryos. Mmp2 mRNA expression is visualized between the 2 myotomes (My), around the neural tube (NT), and below the notochord (NO) where the TD is under formation (circle), in the space between the dorsal aorta (DA) and the posterior-cardinal vein (PCV). YS indicates yolk sac. (B) One-cell transgenic Fli1:eGFPy and Stab1:YFP zebrafish embryos were injected with control (MOctl) or anti-mmp2 (MOmmp2) morpholino oligonucleotides. In 5 dpf control Fli1:eGFPy (top left picture), the TD (white arrow heads) is visible between the DA and the PCV. In control Stab1:YFP zebrafish (bottom left picture), the TD (white arrow heads) is seen above the PCV. Morphant embryos have TD defects (right). (C-D) Quantification of the defective TD formation at 5 dpf. The percentages of embryos with severe (no vessel, 0), drastic (5%-25% of normal TD length), moderate (25%-90% normal TD length), and no (100% normal TD length) lymphatic defects were determined for both transgenic lines after injection of control or anti-mmp2 (MOmmp2) morpholinos. Bars represent 50 μm (A) and 100 μm (B). In panel A, tissue was used mounted in 3% methyl cellulose and images were captured at room temperature with BX60 microscope (Olympus) coupled to DP70 digital camera (Olympus), using acquisition software CellA 3.3 (Olympus Soft Imaging Solutions) and a 40× NA 0.75 VPlan FI lens (Olympus). In panel B, green fluorescent protein or yellow fluorescent protein expressing tissues were used mounted in 3% methyl cellulose and observed immediately under an Eclipse 90i fluorescent microscope (Nikon Instruments). Images were captured at room temperature, with DXM1200C Digital Camera (Nikon Instruments), with acquisition software NIS-Elements 3.00 (Nikon Laboratory Imaging) and a 20× NA 0.5 DIC M/N2 lens (Nikon). Pictures were converted to black and white using Photoshop CS4 software (Adobe Systems).

Mmp2 knockdown impairs TD formation in zebrafish. (A) Mmp2 mRNA expression (purple staining) analyzed by whole-mount in situ hybrization in 3 days postfertilization (dpf) zebrafish embryos. Mmp2 mRNA expression is visualized between the 2 myotomes (My), around the neural tube (NT), and below the notochord (NO) where the TD is under formation (circle), in the space between the dorsal aorta (DA) and the posterior-cardinal vein (PCV). YS indicates yolk sac. (B) One-cell transgenic Fli1:eGFPy and Stab1:YFP zebrafish embryos were injected with control (MOctl) or anti-mmp2 (MOmmp2) morpholino oligonucleotides. In 5 dpf control Fli1:eGFPy (top left picture), the TD (white arrow heads) is visible between the DA and the PCV. In control Stab1:YFP zebrafish (bottom left picture), the TD (white arrow heads) is seen above the PCV. Morphant embryos have TD defects (right). (C-D) Quantification of the defective TD formation at 5 dpf. The percentages of embryos with severe (no vessel, 0), drastic (5%-25% of normal TD length), moderate (25%-90% normal TD length), and no (100% normal TD length) lymphatic defects were determined for both transgenic lines after injection of control or anti-mmp2 (MOmmp2) morpholinos. Bars represent 50 μm (A) and 100 μm (B). In panel A, tissue was used mounted in 3% methyl cellulose and images were captured at room temperature with BX60 microscope (Olympus) coupled to DP70 digital camera (Olympus), using acquisition software CellA 3.3 (Olympus Soft Imaging Solutions) and a 40× NA 0.75 VPlan FI lens (Olympus). In panel B, green fluorescent protein or yellow fluorescent protein expressing tissues were used mounted in 3% methyl cellulose and observed immediately under an Eclipse 90i fluorescent microscope (Nikon Instruments). Images were captured at room temperature, with DXM1200C Digital Camera (Nikon Instruments), with acquisition software NIS-Elements 3.00 (Nikon Laboratory Imaging) and a 20× NA 0.5 DIC M/N2 lens (Nikon). Pictures were converted to black and white using Photoshop CS4 software (Adobe Systems).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal