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FMRP regulates neurogenesis in vivo in Xenopus laevis tadpoles.

Faulkner RL, Wishard TJ, Thompson CK, Liu HH, Cline HT - eNeuro (2015 Jan-Feb)

Bottom Line: Recent studies suggest that loss of FMRP results in aberrant neurogenesis, but neurogenic defects have been variable.Animals with increased or decreased levels of FMRP have significantly decreased neuronal proliferation and survival.These studies show promise in using Xenopus as an experimental system to study fundamental deficits in brain development with loss of FMRP and give new insight into the pathophysiology of FXS.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Dorris Neuroscience Center, Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, California 92037.

ABSTRACT

Fragile X Syndrome (FXS) is the leading known monogenic form of autism and the most common form of inherited intellectual disability. FXS results from silencing the FMR1 gene during embryonic development, leading to loss of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein that regulates mRNA transport, stability, and translation. FXS is commonly thought of as a disease of synaptic dysfunction, however, FMRP expression is lost early in embryonic development, well before most synaptogenesis occurs. Recent studies suggest that loss of FMRP results in aberrant neurogenesis, but neurogenic defects have been variable. We investigated whether FMRP affects neurogenesis in Xenopus laevis tadpoles which express a homolog of FMR1. We used in vivo time-lapse imaging of neural progenitor cells and their neuronal progeny to evaluate the effect of acute loss or over-expression of FMRP on neurogenesis in the developing optic tectum. We complimented the time-lapse studies with SYTOX labeling to quantify apoptosis and CldU labeling to measure cell proliferation. Animals with increased or decreased levels of FMRP have significantly decreased neuronal proliferation and survival. They also have increased neuronal differentiation, but deficient dendritic arbor elaboration. The presence and severity of these defects was highly sensitive to FMRP levels. These data demonstrate that FMRP plays an important role in neurogenesis and suggest that endogenous FMRP levels are carefully regulated. These studies show promise in using Xenopus as an experimental system to study fundamental deficits in brain development with loss of FMRP and give new insight into the pathophysiology of FXS.

No MeSH data available.


Related in: MedlinePlus

FMRP regulates differentiation. In vivo confocal time-lapse images of cells expressing Sox2bd::eGFP and CMO or fmr1a MO collected at 3 dfe and quantification of the changes in neural progenitor cells (NPCs) and neurons over time. A, Confocal Z-projections show the numbers of NPCs (purple arrows), neurons (green arrows), and unidentifiable cells (yellow arrows) in optic tecta expressing CMO, LOW (0.05 mM) fmr1a MO, and HIGH (0.1 mM) fmr1a MO. Dashed lines outline the optic tectum and inset shows a schematic of the optic tectum. B, C, Over 3 d of imaging, there is a decrease in the number of NPCs (B) and an increase in the number of neurons (C) in control animals. LOW and HIGH fmr1a MO decrease the number of NPCs, and HIGH fmr1a MO also decreases the number of neurons (*p < 0.05, **p < 0.01, ***p < 0.001). D, Knockdown of FMRP with HIGH fmr1a MO decreases the proportion of NPCs and increases the proportion of neurons (*p < 0.05). E, Z-projections from in vivo confocal time-lapse images of cells expressing Sox2bd::eGFP + CMO (CMO) or 0.1 mM fmr1a MO (HIGH fmr1a MO), or 1 μg/μl Δfmr1-t2A-eGFP alone (HIGH FMRP OE) or with 0.1 mM fmr1a MO (HIGH MO HIGH Δfmr1 Rescue) at 3 dfe. Dashed lines outline the optic tectum. F, HIGH fmr1a MO and HIGH FMRP OE decrease the number of NPCs and co-electroporation of 1 μg/μl Δfmr1-t2A-eGFP and HIGH fmr1a MO (HIGH MO HIGH Δfmr1 Rescue) partially rescues the defect at 3 dfe with HIGH FMRP OE alone, but does not rescue to control levels (*p < 0.05, **p < 0.01, ***p < 0.001). G, Neuron numbers decrease with HIGH fmr1a MO and this decrease is rescued by co-electroporation of 1 μg/μl Δfmr1-t2A-eGFP (HIGH MO HIGH Δfmr1 Rescue; *p < 0.05). H, HIGH fmr1a MO and HIGH FMRP OE both decrease the proportion of NPCs, and HIGH FMRP OE also increases the proportion of neurons and unidentifiable cells. At 3 dfe, coexpression of HIGH fmr1a MO and 1 μg/μl Δfmr1 partially rescues the HIGH FMRP OE-mediated decrease in NPC proportion, but other defects are not rescued (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bars, 50 μm.
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Figure 6: FMRP regulates differentiation. In vivo confocal time-lapse images of cells expressing Sox2bd::eGFP and CMO or fmr1a MO collected at 3 dfe and quantification of the changes in neural progenitor cells (NPCs) and neurons over time. A, Confocal Z-projections show the numbers of NPCs (purple arrows), neurons (green arrows), and unidentifiable cells (yellow arrows) in optic tecta expressing CMO, LOW (0.05 mM) fmr1a MO, and HIGH (0.1 mM) fmr1a MO. Dashed lines outline the optic tectum and inset shows a schematic of the optic tectum. B, C, Over 3 d of imaging, there is a decrease in the number of NPCs (B) and an increase in the number of neurons (C) in control animals. LOW and HIGH fmr1a MO decrease the number of NPCs, and HIGH fmr1a MO also decreases the number of neurons (*p < 0.05, **p < 0.01, ***p < 0.001). D, Knockdown of FMRP with HIGH fmr1a MO decreases the proportion of NPCs and increases the proportion of neurons (*p < 0.05). E, Z-projections from in vivo confocal time-lapse images of cells expressing Sox2bd::eGFP + CMO (CMO) or 0.1 mM fmr1a MO (HIGH fmr1a MO), or 1 μg/μl Δfmr1-t2A-eGFP alone (HIGH FMRP OE) or with 0.1 mM fmr1a MO (HIGH MO HIGH Δfmr1 Rescue) at 3 dfe. Dashed lines outline the optic tectum. F, HIGH fmr1a MO and HIGH FMRP OE decrease the number of NPCs and co-electroporation of 1 μg/μl Δfmr1-t2A-eGFP and HIGH fmr1a MO (HIGH MO HIGH Δfmr1 Rescue) partially rescues the defect at 3 dfe with HIGH FMRP OE alone, but does not rescue to control levels (*p < 0.05, **p < 0.01, ***p < 0.001). G, Neuron numbers decrease with HIGH fmr1a MO and this decrease is rescued by co-electroporation of 1 μg/μl Δfmr1-t2A-eGFP (HIGH MO HIGH Δfmr1 Rescue; *p < 0.05). H, HIGH fmr1a MO and HIGH FMRP OE both decrease the proportion of NPCs, and HIGH FMRP OE also increases the proportion of neurons and unidentifiable cells. At 3 dfe, coexpression of HIGH fmr1a MO and 1 μg/μl Δfmr1 partially rescues the HIGH FMRP OE-mediated decrease in NPC proportion, but other defects are not rescued (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bars, 50 μm.

Mentions: The experiments described above show that fmr1a MO decreases cell proliferation and survival, however, it is not clear whether one cell type, NPCs or neurons, is more sensitive to FMRP knockdown than another. We therefore investigated whether FMRP knockdown has different effects on the NPCs and neurons within our labeled population. We categorized the labeled cells from the in vivo time-lapse imaging as either NPCs or neurons based on morphology. NPCs are characterized by a triangular cell body and a long radial process extending from the ventricular zone to the pial surface, ending in an elaborated endfoot. Neurons possess a pear-shaped or round soma with elaborated dendritic arbors and an axon. Any cell that lacked a process was categorized as unidentifiable. We quantified the number of NPCs, neurons, and unidentifiable cells to analyze the effect of knockdown on each cell type (Fig. 6A−C). LOW and HIGH fmr1a MO significantly reduced the number of NPCs on all 3 d of imaging compared to CMO (Fig. 6B; 1 dfe NPCs: CMO N = 27 animals; LOW fmr1a MO N = 17 animals, p = 0.027m; HIGH fmr1a MO N = 8 animals, p = 0.011m; 2 dfe NPCs: CMO N = 27 animals; LOW fmr1a MO N = 17 animals, p = 0.030n; HIGH fmr1a MO N = 8 animals, p = 0.0006n; 3 dfe NPCs: CMO N = 27 animals; LOW fmr1a MO N = 17 animals, p = 0.041o; HIGH fmr1a MO N = 8 animals, p = 0.0007o). The reduction of NPC number at 3 dfe was significantly larger for HIGH fmr1a MO compared to LOW fmr1a MO (p = 0.032o). There was a trend toward reduced neuron number with LOW fmr1a MO at 2 and 3 dfe (Fig. 6C; 2 dfe neurons: CMO N = 27 animals; LOW fmr1a MO N = 17 animals, p = 0.080p; 3 dfe neurons: CMO N = 27 animals; LOW fmr1a MO N = 17 animals, p = 0.088q). Combined with the significant decrease in NPCs with LOW fmr1a MO, these results suggest that the increase in cell death detected at 1 dfe in the presence of LOW fmr1a MO may preferentially affect NPCs and nonsignificant reductions in neuron number that appear later are due to depletion of the progenitor pool. HIGH fmr1a MO produced a trend toward reducing neuron number at 2 dfe and significantly reduced the number of neurons at 3 dfe (Fig. 6C; 2 dfe neurons: CMO N = 27 animals; HIGH fmr1a MO N = 8 animals, p = 0.11p; 3 dfe neurons: CMO N = 27 animals; HIGH fmr1a MO N = 8 animals, p = 0.0031q). The decrease in neuron number may be due in part to death of neurons with a higher degree of knockdown, and a decrease in NPC proliferation (Fig. 4C−E) and increased death of NPCs (Fig. 5D,E) likely also contribute to the decreased number of neurons though depletion of the progenitor pool with HIGH fmr1a MO.


FMRP regulates neurogenesis in vivo in Xenopus laevis tadpoles.

Faulkner RL, Wishard TJ, Thompson CK, Liu HH, Cline HT - eNeuro (2015 Jan-Feb)

FMRP regulates differentiation. In vivo confocal time-lapse images of cells expressing Sox2bd::eGFP and CMO or fmr1a MO collected at 3 dfe and quantification of the changes in neural progenitor cells (NPCs) and neurons over time. A, Confocal Z-projections show the numbers of NPCs (purple arrows), neurons (green arrows), and unidentifiable cells (yellow arrows) in optic tecta expressing CMO, LOW (0.05 mM) fmr1a MO, and HIGH (0.1 mM) fmr1a MO. Dashed lines outline the optic tectum and inset shows a schematic of the optic tectum. B, C, Over 3 d of imaging, there is a decrease in the number of NPCs (B) and an increase in the number of neurons (C) in control animals. LOW and HIGH fmr1a MO decrease the number of NPCs, and HIGH fmr1a MO also decreases the number of neurons (*p < 0.05, **p < 0.01, ***p < 0.001). D, Knockdown of FMRP with HIGH fmr1a MO decreases the proportion of NPCs and increases the proportion of neurons (*p < 0.05). E, Z-projections from in vivo confocal time-lapse images of cells expressing Sox2bd::eGFP + CMO (CMO) or 0.1 mM fmr1a MO (HIGH fmr1a MO), or 1 μg/μl Δfmr1-t2A-eGFP alone (HIGH FMRP OE) or with 0.1 mM fmr1a MO (HIGH MO HIGH Δfmr1 Rescue) at 3 dfe. Dashed lines outline the optic tectum. F, HIGH fmr1a MO and HIGH FMRP OE decrease the number of NPCs and co-electroporation of 1 μg/μl Δfmr1-t2A-eGFP and HIGH fmr1a MO (HIGH MO HIGH Δfmr1 Rescue) partially rescues the defect at 3 dfe with HIGH FMRP OE alone, but does not rescue to control levels (*p < 0.05, **p < 0.01, ***p < 0.001). G, Neuron numbers decrease with HIGH fmr1a MO and this decrease is rescued by co-electroporation of 1 μg/μl Δfmr1-t2A-eGFP (HIGH MO HIGH Δfmr1 Rescue; *p < 0.05). H, HIGH fmr1a MO and HIGH FMRP OE both decrease the proportion of NPCs, and HIGH FMRP OE also increases the proportion of neurons and unidentifiable cells. At 3 dfe, coexpression of HIGH fmr1a MO and 1 μg/μl Δfmr1 partially rescues the HIGH FMRP OE-mediated decrease in NPC proportion, but other defects are not rescued (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bars, 50 μm.
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Figure 6: FMRP regulates differentiation. In vivo confocal time-lapse images of cells expressing Sox2bd::eGFP and CMO or fmr1a MO collected at 3 dfe and quantification of the changes in neural progenitor cells (NPCs) and neurons over time. A, Confocal Z-projections show the numbers of NPCs (purple arrows), neurons (green arrows), and unidentifiable cells (yellow arrows) in optic tecta expressing CMO, LOW (0.05 mM) fmr1a MO, and HIGH (0.1 mM) fmr1a MO. Dashed lines outline the optic tectum and inset shows a schematic of the optic tectum. B, C, Over 3 d of imaging, there is a decrease in the number of NPCs (B) and an increase in the number of neurons (C) in control animals. LOW and HIGH fmr1a MO decrease the number of NPCs, and HIGH fmr1a MO also decreases the number of neurons (*p < 0.05, **p < 0.01, ***p < 0.001). D, Knockdown of FMRP with HIGH fmr1a MO decreases the proportion of NPCs and increases the proportion of neurons (*p < 0.05). E, Z-projections from in vivo confocal time-lapse images of cells expressing Sox2bd::eGFP + CMO (CMO) or 0.1 mM fmr1a MO (HIGH fmr1a MO), or 1 μg/μl Δfmr1-t2A-eGFP alone (HIGH FMRP OE) or with 0.1 mM fmr1a MO (HIGH MO HIGH Δfmr1 Rescue) at 3 dfe. Dashed lines outline the optic tectum. F, HIGH fmr1a MO and HIGH FMRP OE decrease the number of NPCs and co-electroporation of 1 μg/μl Δfmr1-t2A-eGFP and HIGH fmr1a MO (HIGH MO HIGH Δfmr1 Rescue) partially rescues the defect at 3 dfe with HIGH FMRP OE alone, but does not rescue to control levels (*p < 0.05, **p < 0.01, ***p < 0.001). G, Neuron numbers decrease with HIGH fmr1a MO and this decrease is rescued by co-electroporation of 1 μg/μl Δfmr1-t2A-eGFP (HIGH MO HIGH Δfmr1 Rescue; *p < 0.05). H, HIGH fmr1a MO and HIGH FMRP OE both decrease the proportion of NPCs, and HIGH FMRP OE also increases the proportion of neurons and unidentifiable cells. At 3 dfe, coexpression of HIGH fmr1a MO and 1 μg/μl Δfmr1 partially rescues the HIGH FMRP OE-mediated decrease in NPC proportion, but other defects are not rescued (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bars, 50 μm.
Mentions: The experiments described above show that fmr1a MO decreases cell proliferation and survival, however, it is not clear whether one cell type, NPCs or neurons, is more sensitive to FMRP knockdown than another. We therefore investigated whether FMRP knockdown has different effects on the NPCs and neurons within our labeled population. We categorized the labeled cells from the in vivo time-lapse imaging as either NPCs or neurons based on morphology. NPCs are characterized by a triangular cell body and a long radial process extending from the ventricular zone to the pial surface, ending in an elaborated endfoot. Neurons possess a pear-shaped or round soma with elaborated dendritic arbors and an axon. Any cell that lacked a process was categorized as unidentifiable. We quantified the number of NPCs, neurons, and unidentifiable cells to analyze the effect of knockdown on each cell type (Fig. 6A−C). LOW and HIGH fmr1a MO significantly reduced the number of NPCs on all 3 d of imaging compared to CMO (Fig. 6B; 1 dfe NPCs: CMO N = 27 animals; LOW fmr1a MO N = 17 animals, p = 0.027m; HIGH fmr1a MO N = 8 animals, p = 0.011m; 2 dfe NPCs: CMO N = 27 animals; LOW fmr1a MO N = 17 animals, p = 0.030n; HIGH fmr1a MO N = 8 animals, p = 0.0006n; 3 dfe NPCs: CMO N = 27 animals; LOW fmr1a MO N = 17 animals, p = 0.041o; HIGH fmr1a MO N = 8 animals, p = 0.0007o). The reduction of NPC number at 3 dfe was significantly larger for HIGH fmr1a MO compared to LOW fmr1a MO (p = 0.032o). There was a trend toward reduced neuron number with LOW fmr1a MO at 2 and 3 dfe (Fig. 6C; 2 dfe neurons: CMO N = 27 animals; LOW fmr1a MO N = 17 animals, p = 0.080p; 3 dfe neurons: CMO N = 27 animals; LOW fmr1a MO N = 17 animals, p = 0.088q). Combined with the significant decrease in NPCs with LOW fmr1a MO, these results suggest that the increase in cell death detected at 1 dfe in the presence of LOW fmr1a MO may preferentially affect NPCs and nonsignificant reductions in neuron number that appear later are due to depletion of the progenitor pool. HIGH fmr1a MO produced a trend toward reducing neuron number at 2 dfe and significantly reduced the number of neurons at 3 dfe (Fig. 6C; 2 dfe neurons: CMO N = 27 animals; HIGH fmr1a MO N = 8 animals, p = 0.11p; 3 dfe neurons: CMO N = 27 animals; HIGH fmr1a MO N = 8 animals, p = 0.0031q). The decrease in neuron number may be due in part to death of neurons with a higher degree of knockdown, and a decrease in NPC proliferation (Fig. 4C−E) and increased death of NPCs (Fig. 5D,E) likely also contribute to the decreased number of neurons though depletion of the progenitor pool with HIGH fmr1a MO.

Bottom Line: Recent studies suggest that loss of FMRP results in aberrant neurogenesis, but neurogenic defects have been variable.Animals with increased or decreased levels of FMRP have significantly decreased neuronal proliferation and survival.These studies show promise in using Xenopus as an experimental system to study fundamental deficits in brain development with loss of FMRP and give new insight into the pathophysiology of FXS.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Dorris Neuroscience Center, Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, California 92037.

ABSTRACT

Fragile X Syndrome (FXS) is the leading known monogenic form of autism and the most common form of inherited intellectual disability. FXS results from silencing the FMR1 gene during embryonic development, leading to loss of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein that regulates mRNA transport, stability, and translation. FXS is commonly thought of as a disease of synaptic dysfunction, however, FMRP expression is lost early in embryonic development, well before most synaptogenesis occurs. Recent studies suggest that loss of FMRP results in aberrant neurogenesis, but neurogenic defects have been variable. We investigated whether FMRP affects neurogenesis in Xenopus laevis tadpoles which express a homolog of FMR1. We used in vivo time-lapse imaging of neural progenitor cells and their neuronal progeny to evaluate the effect of acute loss or over-expression of FMRP on neurogenesis in the developing optic tectum. We complimented the time-lapse studies with SYTOX labeling to quantify apoptosis and CldU labeling to measure cell proliferation. Animals with increased or decreased levels of FMRP have significantly decreased neuronal proliferation and survival. They also have increased neuronal differentiation, but deficient dendritic arbor elaboration. The presence and severity of these defects was highly sensitive to FMRP levels. These data demonstrate that FMRP plays an important role in neurogenesis and suggest that endogenous FMRP levels are carefully regulated. These studies show promise in using Xenopus as an experimental system to study fundamental deficits in brain development with loss of FMRP and give new insight into the pathophysiology of FXS.

No MeSH data available.


Related in: MedlinePlus