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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

The β-actin 3’UTR is sufficient for local translation of Kaede in RGC growth cones in vivo.(a) Steps for in vivo timelapse assay: 1- cDNA injections, 2- selection of embryos with strong Kaede expression in the eye at 2dpf, 3- dissections to remove right eye and drain yolk, 4- photoconversion of Kaede in RGC axons, 5- timelapse. (b) Confocal projection (40x water lens) of a live 3 dpf embryo with Kaede expression in RGC axons in the optic tract (green). (c-f) Confocal projections of the green and red channels of one axon before (c, d) and after photoconversion (e, f). (g-n) Confocal projection from timelapse of one–UTR axon (g-j) and one +UTR axon (k-n) with green to red fluorescence ratio represented by color map. (o, p) Average green to red fluorescence intensity ratio throughout timelapse in growth cones (pixels 1–10) and proximal regions (pixels 141–150) of–UTR (n = 6) and +UTR (n = 10) axons. A one-way ANOVA (p = 0.0012) with a Tukey HSD test (p<0.05) 90 min after photoconversion showed significantly higher green to red ratio in +UTR growth cones compared to–UTR growth cones at 90 min after photoconversion. (q) The green-to-red ratio in a representative +UTR axon (axon 1) plotted against the distance from the growth cone at a representative time (90 min) after photoconversion. The slope of a linear regression to these data (outlined with a magenta rectangle) reflects the spatial gradient of green-to-red ratio 90 minutes after photoconversion. (r) Change in spatial gradient of green-to-red ratio in representative axon 1 throughout the timelapse. The point labeled Q is the slope of the linear regression in panel q, which was determined at 90 min. The other points in this graph were similarly determined in axon 1 at 10 minute intervals throughout the timelapse assay. The slope of a linear regression to these data (outlined with a green rectangle) reflects the rate of change of the gradient of green-to-red fluorescence along axon 1. (s) Rates of change in gradients of green-to-red fluorescence in all assayed axons. The point labeled R is the slope of the linear regression in panel r, which was determined in axon 1. The rates of change of the axonal gradient for the −UTR (n = 6) and +UTR (n = 10) axons were significantly different (Mann-Whitney U test, p = 0.0002). Scale bars are 100 μm (b) and 5 μm (g).
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pone.0134751.g001: The β-actin 3’UTR is sufficient for local translation of Kaede in RGC growth cones in vivo.(a) Steps for in vivo timelapse assay: 1- cDNA injections, 2- selection of embryos with strong Kaede expression in the eye at 2dpf, 3- dissections to remove right eye and drain yolk, 4- photoconversion of Kaede in RGC axons, 5- timelapse. (b) Confocal projection (40x water lens) of a live 3 dpf embryo with Kaede expression in RGC axons in the optic tract (green). (c-f) Confocal projections of the green and red channels of one axon before (c, d) and after photoconversion (e, f). (g-n) Confocal projection from timelapse of one–UTR axon (g-j) and one +UTR axon (k-n) with green to red fluorescence ratio represented by color map. (o, p) Average green to red fluorescence intensity ratio throughout timelapse in growth cones (pixels 1–10) and proximal regions (pixels 141–150) of–UTR (n = 6) and +UTR (n = 10) axons. A one-way ANOVA (p = 0.0012) with a Tukey HSD test (p<0.05) 90 min after photoconversion showed significantly higher green to red ratio in +UTR growth cones compared to–UTR growth cones at 90 min after photoconversion. (q) The green-to-red ratio in a representative +UTR axon (axon 1) plotted against the distance from the growth cone at a representative time (90 min) after photoconversion. The slope of a linear regression to these data (outlined with a magenta rectangle) reflects the spatial gradient of green-to-red ratio 90 minutes after photoconversion. (r) Change in spatial gradient of green-to-red ratio in representative axon 1 throughout the timelapse. The point labeled Q is the slope of the linear regression in panel q, which was determined at 90 min. The other points in this graph were similarly determined in axon 1 at 10 minute intervals throughout the timelapse assay. The slope of a linear regression to these data (outlined with a green rectangle) reflects the rate of change of the gradient of green-to-red fluorescence along axon 1. (s) Rates of change in gradients of green-to-red fluorescence in all assayed axons. The point labeled R is the slope of the linear regression in panel r, which was determined in axon 1. The rates of change of the axonal gradient for the −UTR (n = 6) and +UTR (n = 10) axons were significantly different (Mann-Whitney U test, p = 0.0002). Scale bars are 100 μm (b) and 5 μm (g).

Mentions: Competing Interests: JM, Chief Scientific Officer of Sfidia BioLogic, served as a visiting faculty scientist in CBC’s lab and provided intellectual contribution to the quantification of the Kaede timelapse experiment in Fig 1 of this manuscript. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials, including but not limited to Sfidia BioLogic’s employment, consultancy, patents, or products in development or marketing.


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)

The β-actin 3’UTR is sufficient for local translation of Kaede in RGC growth cones in vivo.(a) Steps for in vivo timelapse assay: 1- cDNA injections, 2- selection of embryos with strong Kaede expression in the eye at 2dpf, 3- dissections to remove right eye and drain yolk, 4- photoconversion of Kaede in RGC axons, 5- timelapse. (b) Confocal projection (40x water lens) of a live 3 dpf embryo with Kaede expression in RGC axons in the optic tract (green). (c-f) Confocal projections of the green and red channels of one axon before (c, d) and after photoconversion (e, f). (g-n) Confocal projection from timelapse of one–UTR axon (g-j) and one +UTR axon (k-n) with green to red fluorescence ratio represented by color map. (o, p) Average green to red fluorescence intensity ratio throughout timelapse in growth cones (pixels 1–10) and proximal regions (pixels 141–150) of–UTR (n = 6) and +UTR (n = 10) axons. A one-way ANOVA (p = 0.0012) with a Tukey HSD test (p<0.05) 90 min after photoconversion showed significantly higher green to red ratio in +UTR growth cones compared to–UTR growth cones at 90 min after photoconversion. (q) The green-to-red ratio in a representative +UTR axon (axon 1) plotted against the distance from the growth cone at a representative time (90 min) after photoconversion. The slope of a linear regression to these data (outlined with a magenta rectangle) reflects the spatial gradient of green-to-red ratio 90 minutes after photoconversion. (r) Change in spatial gradient of green-to-red ratio in representative axon 1 throughout the timelapse. The point labeled Q is the slope of the linear regression in panel q, which was determined at 90 min. The other points in this graph were similarly determined in axon 1 at 10 minute intervals throughout the timelapse assay. The slope of a linear regression to these data (outlined with a green rectangle) reflects the rate of change of the gradient of green-to-red fluorescence along axon 1. (s) Rates of change in gradients of green-to-red fluorescence in all assayed axons. The point labeled R is the slope of the linear regression in panel r, which was determined in axon 1. The rates of change of the axonal gradient for the −UTR (n = 6) and +UTR (n = 10) axons were significantly different (Mann-Whitney U test, p = 0.0002). Scale bars are 100 μm (b) and 5 μm (g).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
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pone.0134751.g001: The β-actin 3’UTR is sufficient for local translation of Kaede in RGC growth cones in vivo.(a) Steps for in vivo timelapse assay: 1- cDNA injections, 2- selection of embryos with strong Kaede expression in the eye at 2dpf, 3- dissections to remove right eye and drain yolk, 4- photoconversion of Kaede in RGC axons, 5- timelapse. (b) Confocal projection (40x water lens) of a live 3 dpf embryo with Kaede expression in RGC axons in the optic tract (green). (c-f) Confocal projections of the green and red channels of one axon before (c, d) and after photoconversion (e, f). (g-n) Confocal projection from timelapse of one–UTR axon (g-j) and one +UTR axon (k-n) with green to red fluorescence ratio represented by color map. (o, p) Average green to red fluorescence intensity ratio throughout timelapse in growth cones (pixels 1–10) and proximal regions (pixels 141–150) of–UTR (n = 6) and +UTR (n = 10) axons. A one-way ANOVA (p = 0.0012) with a Tukey HSD test (p<0.05) 90 min after photoconversion showed significantly higher green to red ratio in +UTR growth cones compared to–UTR growth cones at 90 min after photoconversion. (q) The green-to-red ratio in a representative +UTR axon (axon 1) plotted against the distance from the growth cone at a representative time (90 min) after photoconversion. The slope of a linear regression to these data (outlined with a magenta rectangle) reflects the spatial gradient of green-to-red ratio 90 minutes after photoconversion. (r) Change in spatial gradient of green-to-red ratio in representative axon 1 throughout the timelapse. The point labeled Q is the slope of the linear regression in panel q, which was determined at 90 min. The other points in this graph were similarly determined in axon 1 at 10 minute intervals throughout the timelapse assay. The slope of a linear regression to these data (outlined with a green rectangle) reflects the rate of change of the gradient of green-to-red fluorescence along axon 1. (s) Rates of change in gradients of green-to-red fluorescence in all assayed axons. The point labeled R is the slope of the linear regression in panel r, which was determined in axon 1. The rates of change of the axonal gradient for the −UTR (n = 6) and +UTR (n = 10) axons were significantly different (Mann-Whitney U test, p = 0.0002). Scale bars are 100 μm (b) and 5 μm (g).
Mentions: Competing Interests: JM, Chief Scientific Officer of Sfidia BioLogic, served as a visiting faculty scientist in CBC’s lab and provided intellectual contribution to the quantification of the Kaede timelapse experiment in Fig 1 of this manuscript. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials, including but not limited to Sfidia BioLogic’s employment, consultancy, patents, or products in development or marketing.

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