Journal: Development (Cambridge, England)

Article Title: The parallel growth of motoneuron axons with the dorsal aorta depends on Vegfc/Vegfr3 signaling in zebrafish

doi: 10.1242/dev.091702

Figure Lengend Snippet: Axons of motoneurons develop beneath the dorsal aorta. ( A ) 3D-rendered confocal stack of fluorescence images (lateral view) of an Tg(fli1a;myr-mcherry);(mnx1:gfp) ml2 embryo at 4 dpf. Left column, merged images; center column, GFP images; right column, mCherry images. Lower panels are enlarged images of the boxed regions in the upper panels. Anterior is to the left. Arrows indicate the branch of the descending axon of motoneurons. Arrowheads indicate the extension of axons beneath the DA. ( B ) Left, a single scan confocal image from the stack shown in A. Right, a cross-section image of the stacked image shown in A at the level of the dashed line in the left panel. Arrow denotes the single fascicle of axons. ( C ) 3D-rendered confocal time-sequential stack images of an Tg (fli1a:myr-mcherry);(mnx1:gfp) ml2 embryo. Elapsed time (hours) from the start point of time-lapse imaging (3 dpf) is indicated. Top panels, GFP images; middle panels, merged images of GFP and mCherry; bottom panels, enlarged images of boxed regions of the middle panels. Arrows indicate the tip of a neuronal axon. Arrowheads denote the location of the tip when starting time-lapse imaging. ( D ) Parallel growth (PG) of motoneuron axons with the DA was quantitatively analyzed. ‘Complete’ indicates the complete continuity of the axon between the region above the rostral part of the yolk tube and that above the caudal part of the yolk tube. The number of embryos observed is indicated at the top. ( E ) Single-cell labeling experiment of an embryo of the indicated genotype. Left, 3D-rendered confocal merged image of mCherry and EGFP with a cross-section image at the level of the dashed line. Right, EGFP image with a cross-section image of the EGFP image at the level of the dashed line. Arrows and arrowheads denote the cell bodies of motoneurons in the neural tube and their axons extending beneath the DA, respectively. Scale bars: 25 μm. DA, dorsal aorta; NT, neural tube; PCV, posterior cardinal vein.

Article Snippet: Tg(mnx1:gfp) ml2 fish were obtained from the Zebrafish International Resource Center (University of Oregon, OR, USA).

Techniques: Fluorescence, Imaging, Labeling

Journal: Development (Cambridge, England)

Article Title: The parallel growth of motoneuron axons with the dorsal aorta depends on Vegfc/Vegfr3 signaling in zebrafish

doi: 10.1242/dev.091702

Figure Lengend Snippet: Motoneurons express Vegfr3. ( A ) 3D-rendered confocal stack of fluorescence images (lateral view) of a Tg(fli1a:myr-mcherry);(mnx1:gfp) ml2 embryo at the times indicated in the top panels. Top panels, blood vessels and lymphatic vessels marked by mCherry; middle panels, merged images of GFP and mCherry; bottom panels, motoneurons marked by GFP. Arrows indicate the TD. Dashed lines in the middle panel indicate the outline of the TD. Note that the axon extension preceded TD development and that the axons were formed between DA and TD. Scale bar: 25 μm. ( B ) In situ hybridization analyses of vegfc mRNA in embryos at the indicated stages. Arrows and arrowheads indicate vegfc mRNA detected in the DA in the lateral views (left) and cross-section views (right), respectively. ( C ) RT-PCR analyses using the primers indicated at the left and RNAs prepared from the Tg(fli1a:myr-mcherry);(mnx1:gfp) ml2 embryos by FACS at 72 hpf. ( D ) Real-time quantitative RT-PCR analyses using RNAs prepared from embryos at 72 hpf by the primers as indicated at the bottom. † P <0.05. Error bars indicate s.d. DA, dorsal aorta; EC, endothelial cells; MN, motoneurons; N, notochord; NT, neural tube; PCV, posterior cardinal vein; TD, thoracic duct.

Article Snippet: Tg(mnx1:gfp) ml2 fish were obtained from the Zebrafish International Resource Center (University of Oregon, OR, USA).

Techniques: Fluorescence, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Development (Cambridge, England)

Article Title: The parallel growth of motoneuron axons with the dorsal aorta depends on Vegfc/Vegfr3 signaling in zebrafish

doi: 10.1242/dev.091702

Figure Lengend Snippet: Motoneurons extend neurites in response to recombinant VEGFC. ( A ) Schematic of an ex vivo assay for examining neurite sprouting from motoneurons. Briefly, the Tg(mnx1:gfp) ml2 embryos were cut at the dorsoventral line between the main yolk and the rostrally extended yolk tube and was embedded in matrigel with or without recombinant human vascular endothelial growth factor-C, (h)VEGFC. ( B ) z -stack confocal images (left panel, merged image of transmitted light image and GFP image; center, enlarged image of the boxed region in the left panel; right, GFP image of the boxed region in the left panel) of Tg(mnx1:gfp) ml2 embryos incubated in matrigel. Upper panels, embryo in matrigel without (h)VEGFC; lower four panels, embryos in matrigel with (h)VEGFC and treated as indicated at the left. Red arrows indicate GFP-positive neurites from motoneurons. Anterior is at the top and dorsal to the right. ( C ) Quantitative analyses (ANOVA) of neurite number of the embryos grouped as in B. * P <0.05. Plotted is mean ± s.d. of more than ten embryos for each group. ( D ) Immunoblot analyses with the antibodies indicated at the left using lysates of parental HEK293 and those stably transfected with pPBbsr2(z)vegfr3-f2amcherry and treated with or without (h)VEGFC. A representative result of three independent experiments is shown.

Article Snippet: Tg(mnx1:gfp) ml2 fish were obtained from the Zebrafish International Resource Center (University of Oregon, OR, USA).

Techniques: Recombinant, Ex Vivo, Incubation, Western Blot, Stable Transfection, Transfection

Journal: Development (Cambridge, England)

Article Title: The parallel growth of motoneuron axons with the dorsal aorta depends on Vegfc/Vegfr3 signaling in zebrafish

doi: 10.1242/dev.091702

Figure Lengend Snippet: Overexpression of either Vegfc in the dorsal aorta or Vegfr3 in the motoneurons enhances axon branches of motoneurons over the dorsal aorta. ( A-D ) 3D-rendered confocal stack images (lateral view) of Tg(mnx1:gfp) ml2 embryos transiently expressing the molecules indicated at the bottom left of the left-hand panels. The embryos were intravascularly injected with Qdot 655. Left panels, merged images of GFP and Qdot 655 images; center panels, enlarged image of boxed region of the left panels; right panels, enlarged image of boxed region of the left panels showing GFP signal only. (A) Tg(mnx1:gfp) ml2 embryo transiently expressing mCherry in arteries under the control of the flt1 promoter by Tol2-mediated gene transfer. Scale bar: 25 μm. (B) Zebrafish expressing (z)Vegfc similar to A. (C) Embryo transiently expressing mCherry in motoneurons under the control of the mnx2b promoter by Tol2-mediated gene transfer. (D) Embryo transiently expressing zebrafish (z)Vegfr3 tagged with Flag (F) followed by 2A peptide and mCherry [(z)Vegfr3-F2AmCherry] similar to C. Arrows indicate branches of motoneurons. ( E ) Quantitative analyses of branching of axons over the DA. The number of the embryos showing the increased, normal or decreased parallel growth (PG) divided by the total number ( n ) of embryos counted (indicated at the top) is expressed as percentage of embryos.

Article Snippet: Tg(mnx1:gfp) ml2 fish were obtained from the Zebrafish International Resource Center (University of Oregon, OR, USA).

Techniques: Over Expression, Expressing, Injection, Control

Journal: Development (Cambridge, England)

Article Title: The parallel growth of motoneuron axons with the dorsal aorta depends on Vegfc/Vegfr3 signaling in zebrafish

doi: 10.1242/dev.091702

Figure Lengend Snippet: Requirement of Vegfc and Vegfr3 for alignment of motoneuron axons and dorsal aorta. ( A ) 3D-rendered confocal stack images (lateral view) of Tg(fli1a:myr-mcherry);(mnx1:gfp) ml2 embryos treated with control DMSO (Ctrl, upper panels), with maz51 (middle panels) or with ki8751 (lower panels) at 4 dpf. Left panels, merged images of mCherry and GFP; right panels, GFP images. White arrows indicate the motoneurons beneath the DA. Arrowheads indicate the reduced growth of motoneuron axons beneath the DA. Yellow arrows indicate the slight reduction of growth of motoneuron axon beneath the DA. ( B ) 3D-rendered confocal stack images (lateral view) of Tg(fli1a:myr-mcherry);(mnx1:gfp) ml2 embryos treated with control MO (Ctrl, top panels), with vegfc MO (middle panels) or with vegfr3 MO (bottom panels). Left panels, merged images of mCherry and GFP; right panels, GFP images. Arrows indicate the motoneuron axons beneath the DA. Arrowheads indicate the reduced interaction of motoneurons and dorsal aorta. ( C ) Quantitative analyses of impairment of parallel growth (PG) of the embryos grouped as in B. The number ( n ) of the embryos examined for quantitative analyses is indicated at the top. ( D ) Immunohistochemical study of Tg(fli1a:egfp) y1 embryos (top) and vegfr3 hu4602/wt ( expando mutant); Tg(fli1a:egfp) y1 embryos using anti-znp1 antibody. Arrowheads and arrows indicate the presence or absence of axons beneath the dorsal aorta, respectively. Note that the TD is absent in the expando mutant. Scale bar: 25 μm. DA, dorsal aorta; TD, thoracic duct.

Article Snippet: Tg(mnx1:gfp) ml2 fish were obtained from the Zebrafish International Resource Center (University of Oregon, OR, USA).

Techniques: Control, Immunohistochemical staining, Mutagenesis

Journal: Development (Cambridge, England)

Article Title: The parallel growth of motoneuron axons with the dorsal aorta depends on Vegfc/Vegfr3 signaling in zebrafish

doi: 10.1242/dev.091702

Figure Lengend Snippet: Inhibition of Vegfr3 in the motoneuron results in impairment of alignment of the motoneuron axons and dorsal aorta. ( A ) 3D-rendered confocal stack images (lateral view) of Tg(fli1a:myr-mcherry);(mnx1:gfp) ml2 embryos (upper panel) and those transiently expressing (h)VEGFR3-Fc in the motoneurons under the control of the mnx2b promoter by Tol2-mediated gene transfer (lower panels). Left panels, merged images of mCherry and GFP; right panels, GFP images. Arrows indicate the motoneuron axon beneath the dorsal aorta (DA). Arrowheads indicate the impairment of parallel growth of motoneuron axon with the DA. Scale bar: 25 μm. ( B ) Quantitative analyses of the impaired parallel growth (PG) of the embryos grouped as in A. The number ( n ) of the embryos observed indicated at the top.

Article Snippet: Tg(mnx1:gfp) ml2 fish were obtained from the Zebrafish International Resource Center (University of Oregon, OR, USA).

Techniques: Inhibition, Expressing, Control