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The RNA Binding Protein Igf2bp1 Is Required for Zebrafish RGC Axon Outgrowth In Vivo.

Gaynes JA, Otsuna H, Campbell DS, Manfredi JP, Levine EM, Chien CB - PLoS ONE (2015)

Bottom Line: Igf2bp1 knockdown reduced RGC axonal outgrowth and tectal coverage and retinal cell survival.RGC-specific expression of a phosphomimetic Igf2bp1 reduced the density of axonal projections in the optic tract while sparing RGCs, demonstrating for the first time that Igf2bp1 is required during axon outgrowth in vivo.Therefore, regulation of local translation mediated by Igf2bp proteins may be required at all stages of axon development.

View Article: PubMed Central - PubMed

Affiliation: Program in Neuroscience, University of Utah Medical Center, Salt Lake City, Utah, United States of America; Department of Neurobiology and Anatomy, University of Utah Medical Center, Salt Lake City, Utah, United States of America; Department of Ophthalmology/Visual Sciences, John A. Moran Center, University of Utah Medical Center, Salt Lake City, Utah, United States of America.

ABSTRACT
Attractive growth cone turning requires Igf2bp1-dependent local translation of β-actin mRNA in response to external cues in vitro. While in vivo studies have shown that Igf2bp1 is required for cell migration and axon terminal branching, a requirement for Igf2bp1 function during axon outgrowth has not been demonstrated. Using a timelapse assay in the zebrafish retinotectal system, we demonstrate that the β-actin 3'UTR is sufficient to target local translation of the photoconvertible fluorescent protein Kaede in growth cones of pathfinding retinal ganglion cells (RGCs) in vivo. Igf2bp1 knockdown reduced RGC axonal outgrowth and tectal coverage and retinal cell survival. RGC-specific expression of a phosphomimetic Igf2bp1 reduced the density of axonal projections in the optic tract while sparing RGCs, demonstrating for the first time that Igf2bp1 is required during axon outgrowth in vivo. Therefore, regulation of local translation mediated by Igf2bp proteins may be required at all stages of axon development.

No MeSH data available.


Related in: MedlinePlus

Impairment of Igf2bp1 perturbs retinotectal projections.(a) The e3i3 MO targeted to the e3i3 splice junction in Igf2bp1 pre-mRNA. (b) Left gel: forward primer targeted to exon 1 in the 5’UTR (Fprimer) and a reverse primer targeted to exon 3 (Rprimer1) yielded no detectable product as expected with exon 3 deletion. Right gel: Fprimer and a reverse primer targeted to exon 4 (Rprimer2) yielded a PCR product that was TOPO-TA cloned and sequenced (c) to verify exon 3 deletion (blank lanes were cropped out of gel image), which results in a severely truncated protein (d). (e-n) Transmitted light images of whole 3 dpf control (e,f) and e3i3 MO-injected morphants (g-i) and head regions (j-n) with eyes (yellow outline) and hearts (blue arrowheads). (o-s) 3D projections made from confocal z-stacks taken with a 30x silicone immersion lens on a confocal microscope, of Tg(isl2b:mCherryCAAX)zc23 3 dpf embryos stained with α-DsRed (red) and counterstained with TO-PRO-3 (blue), with one example each for uninjected (o), or injected with negative control MO (p), e3i3 MO (mild (q), moderate (r), severe (s)), reconstructed from confocal z-stacks with FluoRender software, rotated to give lateral (top panels), coronal (middle panels) and dorsal (bottom panels) views. Scale bar is 100 μm.
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pone.0134751.g003: Impairment of Igf2bp1 perturbs retinotectal projections.(a) The e3i3 MO targeted to the e3i3 splice junction in Igf2bp1 pre-mRNA. (b) Left gel: forward primer targeted to exon 1 in the 5’UTR (Fprimer) and a reverse primer targeted to exon 3 (Rprimer1) yielded no detectable product as expected with exon 3 deletion. Right gel: Fprimer and a reverse primer targeted to exon 4 (Rprimer2) yielded a PCR product that was TOPO-TA cloned and sequenced (c) to verify exon 3 deletion (blank lanes were cropped out of gel image), which results in a severely truncated protein (d). (e-n) Transmitted light images of whole 3 dpf control (e,f) and e3i3 MO-injected morphants (g-i) and head regions (j-n) with eyes (yellow outline) and hearts (blue arrowheads). (o-s) 3D projections made from confocal z-stacks taken with a 30x silicone immersion lens on a confocal microscope, of Tg(isl2b:mCherryCAAX)zc23 3 dpf embryos stained with α-DsRed (red) and counterstained with TO-PRO-3 (blue), with one example each for uninjected (o), or injected with negative control MO (p), e3i3 MO (mild (q), moderate (r), severe (s)), reconstructed from confocal z-stacks with FluoRender software, rotated to give lateral (top panels), coronal (middle panels) and dorsal (bottom panels) views. Scale bar is 100 μm.

Mentions: We hypothesized that loss of Igf2bp1 function would cause RGC pathfinding errors. 1-cell embryos were injected with a splice-blocking MO, targeted against the exon3-intron3 splice junction (e3i3) (Fig 3A). A series of injections ranging from 1–12 ng revealed that 3 ng was the maximal dose with minimal effects on organism survival at 3 dpf (data not shown). The effect of a 3 ng injection on endogenous Igf2bp1 mRNA transcript was determined by RT-PCR using RNA from injected embryos at 2 dpf, which revealed a 61 bp deletion corresponding to exon 3 (Fig 3A–3D). This deletion is predicted to cause a frame shift at amino acid 81 and an early in-frame stop codon after amino acid 91 (Fig 3D), yielding a severely truncated protein that would be unable to bind β-actin mRNA. Therefore, we conclude that e3i3 MO effectively impairs endogenous Igf2bp1 transcript.


The RNA Binding Protein Igf2bp1 Is Required for Zebrafish RGC Axon Outgrowth In Vivo.

Gaynes JA, Otsuna H, Campbell DS, Manfredi JP, Levine EM, Chien CB - PLoS ONE (2015)

Impairment of Igf2bp1 perturbs retinotectal projections.(a) The e3i3 MO targeted to the e3i3 splice junction in Igf2bp1 pre-mRNA. (b) Left gel: forward primer targeted to exon 1 in the 5’UTR (Fprimer) and a reverse primer targeted to exon 3 (Rprimer1) yielded no detectable product as expected with exon 3 deletion. Right gel: Fprimer and a reverse primer targeted to exon 4 (Rprimer2) yielded a PCR product that was TOPO-TA cloned and sequenced (c) to verify exon 3 deletion (blank lanes were cropped out of gel image), which results in a severely truncated protein (d). (e-n) Transmitted light images of whole 3 dpf control (e,f) and e3i3 MO-injected morphants (g-i) and head regions (j-n) with eyes (yellow outline) and hearts (blue arrowheads). (o-s) 3D projections made from confocal z-stacks taken with a 30x silicone immersion lens on a confocal microscope, of Tg(isl2b:mCherryCAAX)zc23 3 dpf embryos stained with α-DsRed (red) and counterstained with TO-PRO-3 (blue), with one example each for uninjected (o), or injected with negative control MO (p), e3i3 MO (mild (q), moderate (r), severe (s)), reconstructed from confocal z-stacks with FluoRender software, rotated to give lateral (top panels), coronal (middle panels) and dorsal (bottom panels) views. Scale bar is 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556669&req=5

pone.0134751.g003: Impairment of Igf2bp1 perturbs retinotectal projections.(a) The e3i3 MO targeted to the e3i3 splice junction in Igf2bp1 pre-mRNA. (b) Left gel: forward primer targeted to exon 1 in the 5’UTR (Fprimer) and a reverse primer targeted to exon 3 (Rprimer1) yielded no detectable product as expected with exon 3 deletion. Right gel: Fprimer and a reverse primer targeted to exon 4 (Rprimer2) yielded a PCR product that was TOPO-TA cloned and sequenced (c) to verify exon 3 deletion (blank lanes were cropped out of gel image), which results in a severely truncated protein (d). (e-n) Transmitted light images of whole 3 dpf control (e,f) and e3i3 MO-injected morphants (g-i) and head regions (j-n) with eyes (yellow outline) and hearts (blue arrowheads). (o-s) 3D projections made from confocal z-stacks taken with a 30x silicone immersion lens on a confocal microscope, of Tg(isl2b:mCherryCAAX)zc23 3 dpf embryos stained with α-DsRed (red) and counterstained with TO-PRO-3 (blue), with one example each for uninjected (o), or injected with negative control MO (p), e3i3 MO (mild (q), moderate (r), severe (s)), reconstructed from confocal z-stacks with FluoRender software, rotated to give lateral (top panels), coronal (middle panels) and dorsal (bottom panels) views. Scale bar is 100 μm.
Mentions: We hypothesized that loss of Igf2bp1 function would cause RGC pathfinding errors. 1-cell embryos were injected with a splice-blocking MO, targeted against the exon3-intron3 splice junction (e3i3) (Fig 3A). A series of injections ranging from 1–12 ng revealed that 3 ng was the maximal dose with minimal effects on organism survival at 3 dpf (data not shown). The effect of a 3 ng injection on endogenous Igf2bp1 mRNA transcript was determined by RT-PCR using RNA from injected embryos at 2 dpf, which revealed a 61 bp deletion corresponding to exon 3 (Fig 3A–3D). This deletion is predicted to cause a frame shift at amino acid 81 and an early in-frame stop codon after amino acid 91 (Fig 3D), yielding a severely truncated protein that would be unable to bind β-actin mRNA. Therefore, we conclude that e3i3 MO effectively impairs endogenous Igf2bp1 transcript.

Bottom Line: Igf2bp1 knockdown reduced RGC axonal outgrowth and tectal coverage and retinal cell survival.RGC-specific expression of a phosphomimetic Igf2bp1 reduced the density of axonal projections in the optic tract while sparing RGCs, demonstrating for the first time that Igf2bp1 is required during axon outgrowth in vivo.Therefore, regulation of local translation mediated by Igf2bp proteins may be required at all stages of axon development.

View Article: PubMed Central - PubMed

Affiliation: Program in Neuroscience, University of Utah Medical Center, Salt Lake City, Utah, United States of America; Department of Neurobiology and Anatomy, University of Utah Medical Center, Salt Lake City, Utah, United States of America; Department of Ophthalmology/Visual Sciences, John A. Moran Center, University of Utah Medical Center, Salt Lake City, Utah, United States of America.

ABSTRACT
Attractive growth cone turning requires Igf2bp1-dependent local translation of β-actin mRNA in response to external cues in vitro. While in vivo studies have shown that Igf2bp1 is required for cell migration and axon terminal branching, a requirement for Igf2bp1 function during axon outgrowth has not been demonstrated. Using a timelapse assay in the zebrafish retinotectal system, we demonstrate that the β-actin 3'UTR is sufficient to target local translation of the photoconvertible fluorescent protein Kaede in growth cones of pathfinding retinal ganglion cells (RGCs) in vivo. Igf2bp1 knockdown reduced RGC axonal outgrowth and tectal coverage and retinal cell survival. RGC-specific expression of a phosphomimetic Igf2bp1 reduced the density of axonal projections in the optic tract while sparing RGCs, demonstrating for the first time that Igf2bp1 is required during axon outgrowth in vivo. Therefore, regulation of local translation mediated by Igf2bp proteins may be required at all stages of axon development.

No MeSH data available.


Related in: MedlinePlus