16 research outputs found
TNFR2 is required for glial ensheathment of DRG axons at the DREZ.
<p>(A) Images of <i>Tg(sox10</i>:<i>eos)</i> wildtype, <i>tnfr2</i> morphant and <i>tnfr2</i> F0 CRISPR mutant larvae at 72 hpf showing a central branch that is ensheathed by <i>sox10</i><sup>+</sup> glia (arrow) in wildtype but not <i>tnfr2</i> morphants or <i>tnfr2</i> F0 CRISPR mutants (arrow). (B) Quantification of data from panel A (wt n = 40 DRG, <i>tnfr2</i> MO n = 31 DRG, <i>tnfr1</i> = 24 DRG). (C) Images of spinal column tissue from wildtype and <i>TNFR2</i><sup><i>-/-</i></sup> P0 mouse embryos labeled with antibodies to S100 (green) and βIII Tubulin (red). Traced area denotes central projection that extends into the spinal cord. (D) Electron microscopy images of DRG tissue from wildtype and <i>TNFR2</i><sup><i>-/-</i></sup> pups showed a reduction in the ensheathment of axonal bundles. (E,F) Quantification of ensheathed vs unensheathed bundles in wildtype and <i>TNFR2</i><sup><i>-/-</i></sup> pups (3 animals, wt n = 108 nerve bundles, <i>TNFR2-/-</i> n = 68 nerve bundles). (G,H) Quantification of the number of axons per small-caliber bundle in wildtype and <i>TNFR2</i><sup><i>-/-</i></sup> pups. (I) Images from a 24-hour time-lapse movie of a <i>Tg(sox10</i>:<i>eos)</i> embryo injected with <i>tnfr2</i> MO. Fisher’s exact test (B,F,H). Students t-test (E,F). Scale bars, 25 μm (A,C,I), 5 μm (D).</p
TNFa is required for axonal ensheathment by DREZ glia.
<p>(A) Images of wildtype, <i>tnfa</i> morphant and <i>tnfa</i> F0 CRISPR mutant <i>Tg(sox10</i>:<i>eos)</i> larvae at 72 hpf. Arrow denotes central branch that is ensheathed by <i>sox10</i><sup>+</sup> glia in wildtype but not <i>tnfa</i> morphants or <i>tnfa</i> F0 CRISPR mutants. (B) Quantification of data from panel A, wt n = 40 DRG, <i>tnfa</i> MO n = 54 DRG. (C) Electron microscopy images of DRG tissue from wildtype and <i>TNF</i><sup><i>-/-</i></sup> P0 pups showed a reduction in the ensheathment of axonal bundles. (n = 3 animals/genotype). (D,E) Quantification of ensheathed vs unensheathed bundles in wildtype and <i>TNF</i><sup><i>-/-</i></sup> pups (3 animals, wt n = 108 nerve bundles, <i>TNFR2-/-</i> n = 85 nerve bundles). (F,G) Quantification of the number of axons per small-caliber bundle in wildtype and <i>TNF</i><sup><i>-/-</i></sup> pups. (H) Images of spinal column tissue from wildtype and <i>TNF</i><sup><i>-/-</i></sup> P0 embryo labeled with antibodies to S100 (green) and βIII Tubulin (red). Traced area denotes central projection that extends into the spinal cord. In <i>TNF</i><sup><i>-/-</i></sup> pups, the central afferent lacks S100 staining. Fisher’s exact test (B,E,G). Students t-test (D,F). Scale bars, 25 μm (A,H), 5 μm (C).</p
DRG glia navigate with pioneer axons.
<p>Images from a 24 hour time-lapse movie starting at 48 hpf in a <i>Tg(sox10</i>:<i>eos);Tg(ngn1</i>:<i>egfp)</i> embryo that was exposed to UV light to photoconvert Eos in the whole embryo. These frames show the pioneer axon (green) and associated glia (red) migrate together to the DREZ. Below are individual channels for GFP and Eos. Arrows denote the pioneer axon growth cone and arrowheads denote location of the DRG glia. Scale bar, 25 μm.</p
TNFa/TNFR2-mediated signaling is active in pioneer axon-associated glia.
<p>(A) Images and schematic of <i>Tg(NFkB</i>:<i>egfp);Tg(sox10</i>:<i>mrfp)</i> embryos at 72 hpf showing GFP<sup>+</sup> glia at the DREZ. (B) Quantification of the percentage of DRG with <i>Tg(NFkB</i>:<i>egfp)</i> expression at 48 and 72 hpf, n = 30 DRG. (C) Quantification of NFkB<sup>+</sup> DREZ glia along pioneer axons that were axotomized before entry (pre-entry), after entry (post-entry), non-ablated and peripheral axotomy (n = 8 nerves/condition). (D) Intensity profiles of GFP in <i>Tg(NFkB</i>:<i>egfp);Tg(sox10</i>:<i>mrfp)</i> embryos from 48 to 72 hpf showing GFP upregulation upon axon entry into the spinal cord. Shown is the average of 2 movies normalized for the time of entry. (E) Images of <i>Tg(NFkB</i>:<i>egfp)</i> embryos in wildtype, <i>tnfa</i> morphant and <i>tnfr2</i> morphant larvae showing NFkB activation is dependent on <i>tnfa</i> and <i>tnfr2</i>. (F) Quantification of data from panel D, SEM is shown, wt n = 30 DRG, <i>tnfa</i> MO n = 30 DRG, <i>tnfr2</i> MO n = 30 DRG. Scale bars, 25 μm.</p
DRG neurons upregulate TNFa after pioneer axons enter the spinal cord.
<p>(A) At 72 hpf, <i>Tg(tnfa</i>:<i>gfp)</i> zebrafish embryos show robust expression of GFP in DRG neurons after the pioneer axon has entered the spinal cord. Arrow demarcates the peripheral projection labeled with acetylated tubulin. (B) Quantification of <i>tnfa</i><sup>+</sup> DRG neurons at 48, 52 and 72 hpf (n = 30 DRG nerves). (C) Excerpt from a 24 hour time-lapse movie starting at 48 hpf in a <i>Tg(tnfa</i>:<i>gfp);Tg(sox10</i>:<i>mrfp)</i> embryo shows GFP expression in DRG neurons increasing as the pioneer axon (arrow), identified by RFP, enters the spinal cord. Arrowhead denotes DRG neuron cell soma. (D) Intensity profile of GFP in movie shown in panel C (n = 7 DRG). (E) Live images of a 48 hpf <i>Tg(tnfa</i>:<i>gfp);Tg(sox10</i>:<i>mrfp)</i> embryo showing a pioneer axon (arrow) just as it has formed pre-axotomy, 1 hpa and 24 hpa. In these images, axotomy prevents pioneer axons from entering the spinal cord and GFP expression is never observed. Arrowhead denotes DRG neuron cell soma. (F) Live images of a 48 hpf <i>Tg(tnfa</i>:<i>gfp);Tg(sox10</i>:<i>mrfp)</i> embryo where the pioneer axon (arrow) had already entered the spinal cord. In this instance, axotomy did not affect GFP expression. (G) Quantification of GFP expression in DRG neurons from panels E and F (n = 5 nerves). Scale bars, 25 μm.</p
DRG pioneer axons navigate directly to the DREZ.
<p>(A) Images and traced schematics from a 24 hour time-lapse movie starting at 48 hpf in a <i>Tg(ngn1</i>:<i>egfp)</i> embryo showing navigation of the DRG pioneer axon toward the DREZ. Orange box indicates location of DREZ. Arrows denote the growth cone and arrowheads denote location of cell body. Blue process in schematic denotes peripheral axon. (B) Quantification of the distance (y-axis) and time (x-axis) of 4 growth cones as they navigated toward the DREZ. (C) Schematic tracings of 3 pioneer axons as they navigated to the DREZ. Compass shows Dorsal-D, Ventral-V, Anterior-A, Posterior-P. Scale bar, 25 μm.</p
AAV-mediated expression of HLA-G for the prevention of experimental ocular graft vs. host disease
Ocular graft versus host disease (OGvHD) develops after allogeneic hematopoietic stem cell transplantation (HSCT) and manifests as ocular surface inflammatory disease. This study evaluated the efficacy of adeno-associated virus (AAV) gene therapy encoding human leukocyte antigen G (HLA-G) to inhibit OGvHD. A major histocompatibility mismatch chronic OGvHD murine model was evaluated. 7Â days after HSCT, mice were dosed subconjunctivally with scAAV8-HLA-G1/5 (1 x 109 vg/eye), topical cyclosporine (twice daily), or left untreated. Body weights and tear production (red thread test) were recorded, and eyelid, corneal opacity, and corneal fluorescein retention were scored through day 44 after HSCT. Tissues were collected for vector biodistribution, ocular histology, and immunofluorescence. Compared with untreated HSCT eyes, those dosed with scAAV8-HLA-G1/5 had significantly reduced clinical inflammatory signs of OGvHD. On histology, eyes that received scAAV8-HLA-G1/5 or cyclosporine had a significantly lower mean limbal mononuclear cell count when compared with non-treated HSCT eyes. HLA-G immunofluorescence was detected in the subconjunctiva and peripheral cornea in HSCT animals treated with scAAV8-HLA-G1/5. Vector genomes were detected in the lacrimal gland, but not in the other tested organs. These results provide evidence that subconjunctival AAV targets ocular surface and corneal disease and support that HLA-G-based gene therapy may be an effective treatment for OGvHD