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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 increases cell death and layering defects in the retina.Tg(isl2b:GFP)zc7 stable transgenic embryos (a-h) were uninjected (a, e) injected with p53 MO (b, f), e3i3 (c, g), or co-injected with p53 MO+e3i3 MO (d, h). Embryos were fixed and stained with α-EGFP (green) and TO-PRO-3 (red in a-d, gray in e-h). Images are maximum intensity projections (a-d, i-l) or singles slices (e-h) of lateral views of eyes, with lens removed, taken with a confocal microscope (20x lens). Morphant eyes were missing RGCs in central retina (white arrowheads in c, d) and had displaced RGCs (yellow arrowheads in c, d). (g, h) Single z-slices have holes (white arrowheads) and abnormal layers (yellow arrowheads). (i, l) Maximum intensity projections, lateral view of AO staining in 31 hpf embryos, within the lens (inner dotted yellow circle) and retina (outer dotted yellow circle). (m) The AO-positive cells were counted in retinas alone (not including lens and extraocular tissues) from; uninjected (n = 10), p53 MO injected (n = 9), e3i3 MO injected (n = 10), and coinjected with p53 MO and e3i3 MO (n = 10) embryos. A one-way ANOVA (p<0.0001) with Tukey HSD test (p<0.01) showed that e3i3 MO injected embryos and embryos coinjected with e3i3 MO and p53 MO were both significantly different than controls, but not significantly different from each other. The black points on the graph represent mean +/- SEM. Scale bars are 50 μm.
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pone.0134751.g004: Impairment of Igf2bp1 increases cell death and layering defects in the retina.Tg(isl2b:GFP)zc7 stable transgenic embryos (a-h) were uninjected (a, e) injected with p53 MO (b, f), e3i3 (c, g), or co-injected with p53 MO+e3i3 MO (d, h). Embryos were fixed and stained with α-EGFP (green) and TO-PRO-3 (red in a-d, gray in e-h). Images are maximum intensity projections (a-d, i-l) or singles slices (e-h) of lateral views of eyes, with lens removed, taken with a confocal microscope (20x lens). Morphant eyes were missing RGCs in central retina (white arrowheads in c, d) and had displaced RGCs (yellow arrowheads in c, d). (g, h) Single z-slices have holes (white arrowheads) and abnormal layers (yellow arrowheads). (i, l) Maximum intensity projections, lateral view of AO staining in 31 hpf embryos, within the lens (inner dotted yellow circle) and retina (outer dotted yellow circle). (m) The AO-positive cells were counted in retinas alone (not including lens and extraocular tissues) from; uninjected (n = 10), p53 MO injected (n = 9), e3i3 MO injected (n = 10), and coinjected with p53 MO and e3i3 MO (n = 10) embryos. A one-way ANOVA (p<0.0001) with Tukey HSD test (p<0.01) showed that e3i3 MO injected embryos and embryos coinjected with e3i3 MO and p53 MO were both significantly different than controls, but not significantly different from each other. The black points on the graph represent mean +/- SEM. Scale bars are 50 μm.

Mentions: We examined morphant retinas to determine if RGC axons were unable to exit the eye (Fig 4A–4D). In order to prevent non-specific cell death [40] we co-injected p53 MO with the e3i3 MO into Tg(isl2b:GFP)zc7 embryos, which express GFP in the majority of RGCs (Fig 4A–4D). RGC axons normally exit the eye through the optic disc in the ventral retina, where netrin is expressed [41]. When we examined confocal images of eyes in morphant embryos we did not see an accumulation of RGC axons and growth cones inside the retinas, as would be expected if RGC axons were stalled or unable to exit the eyes. However, some retinal defects were noted (Fig 4A–4H). The RGC layer was thinner and the border was less defined in morphant retinas, with several displaced GFP+ RGCs (Fig 4C and 4D, yellow arrowheads). Furthermore, there were large holes in the central domains of morphant retinas, where both GFP and TO-PRO-3 staining were absent (Fig 4C and 4D, white arrow heads), suggesting that RGCs were absent. Pioneer RGCs located in the central retina are the first to differentiate, and outgrowth of axons from later born RGCs is dependent on interactions with pioneer axons [37]. Therefore, loss of central RGCs could cause a reduction in the number of axons that exit the eye. There were also small holes seen in the TO-PRO-3 staining in morphant retinas (Fig 4G and 4H, white arrowheads), suggesting that cell death was occurring.


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 increases cell death and layering defects in the retina.Tg(isl2b:GFP)zc7 stable transgenic embryos (a-h) were uninjected (a, e) injected with p53 MO (b, f), e3i3 (c, g), or co-injected with p53 MO+e3i3 MO (d, h). Embryos were fixed and stained with α-EGFP (green) and TO-PRO-3 (red in a-d, gray in e-h). Images are maximum intensity projections (a-d, i-l) or singles slices (e-h) of lateral views of eyes, with lens removed, taken with a confocal microscope (20x lens). Morphant eyes were missing RGCs in central retina (white arrowheads in c, d) and had displaced RGCs (yellow arrowheads in c, d). (g, h) Single z-slices have holes (white arrowheads) and abnormal layers (yellow arrowheads). (i, l) Maximum intensity projections, lateral view of AO staining in 31 hpf embryos, within the lens (inner dotted yellow circle) and retina (outer dotted yellow circle). (m) The AO-positive cells were counted in retinas alone (not including lens and extraocular tissues) from; uninjected (n = 10), p53 MO injected (n = 9), e3i3 MO injected (n = 10), and coinjected with p53 MO and e3i3 MO (n = 10) embryos. A one-way ANOVA (p<0.0001) with Tukey HSD test (p<0.01) showed that e3i3 MO injected embryos and embryos coinjected with e3i3 MO and p53 MO were both significantly different than controls, but not significantly different from each other. The black points on the graph represent mean +/- SEM. Scale bars are 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556669&req=5

pone.0134751.g004: Impairment of Igf2bp1 increases cell death and layering defects in the retina.Tg(isl2b:GFP)zc7 stable transgenic embryos (a-h) were uninjected (a, e) injected with p53 MO (b, f), e3i3 (c, g), or co-injected with p53 MO+e3i3 MO (d, h). Embryos were fixed and stained with α-EGFP (green) and TO-PRO-3 (red in a-d, gray in e-h). Images are maximum intensity projections (a-d, i-l) or singles slices (e-h) of lateral views of eyes, with lens removed, taken with a confocal microscope (20x lens). Morphant eyes were missing RGCs in central retina (white arrowheads in c, d) and had displaced RGCs (yellow arrowheads in c, d). (g, h) Single z-slices have holes (white arrowheads) and abnormal layers (yellow arrowheads). (i, l) Maximum intensity projections, lateral view of AO staining in 31 hpf embryos, within the lens (inner dotted yellow circle) and retina (outer dotted yellow circle). (m) The AO-positive cells were counted in retinas alone (not including lens and extraocular tissues) from; uninjected (n = 10), p53 MO injected (n = 9), e3i3 MO injected (n = 10), and coinjected with p53 MO and e3i3 MO (n = 10) embryos. A one-way ANOVA (p<0.0001) with Tukey HSD test (p<0.01) showed that e3i3 MO injected embryos and embryos coinjected with e3i3 MO and p53 MO were both significantly different than controls, but not significantly different from each other. The black points on the graph represent mean +/- SEM. Scale bars are 50 μm.
Mentions: We examined morphant retinas to determine if RGC axons were unable to exit the eye (Fig 4A–4D). In order to prevent non-specific cell death [40] we co-injected p53 MO with the e3i3 MO into Tg(isl2b:GFP)zc7 embryos, which express GFP in the majority of RGCs (Fig 4A–4D). RGC axons normally exit the eye through the optic disc in the ventral retina, where netrin is expressed [41]. When we examined confocal images of eyes in morphant embryos we did not see an accumulation of RGC axons and growth cones inside the retinas, as would be expected if RGC axons were stalled or unable to exit the eyes. However, some retinal defects were noted (Fig 4A–4H). The RGC layer was thinner and the border was less defined in morphant retinas, with several displaced GFP+ RGCs (Fig 4C and 4D, yellow arrowheads). Furthermore, there were large holes in the central domains of morphant retinas, where both GFP and TO-PRO-3 staining were absent (Fig 4C and 4D, white arrow heads), suggesting that RGCs were absent. Pioneer RGCs located in the central retina are the first to differentiate, and outgrowth of axons from later born RGCs is dependent on interactions with pioneer axons [37]. Therefore, loss of central RGCs could cause a reduction in the number of axons that exit the eye. There were also small holes seen in the TO-PRO-3 staining in morphant retinas (Fig 4G and 4H, white arrowheads), suggesting that cell death was occurring.

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