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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

Knockdown and overexpression of FMRP decrease proliferation. A, Z-projections from in vivo confocal time-lapse images of cells expressing Sox2bd::eGFP + CMO (CMO) or 0.05 mM fmr1a MO (LOW fmr1a MO), and 1 μg/μl Δfmr1-t2A-eGFP + 0.05 mM fmr1a MO (LOW MO HIGH Δfmr1 Rescue) taken at 1 and 3 dfe. Dashed lines outline the optic tectum and inset shows a schematic of the optic tectum. B, The percent change in the number of eGFP+ cells increases over 3 d in CMO animals. FMRP knockdown with LOW fmr1a MO blocks the increase in cell number between 1 − 3 dfe. Coexpression of LOW fmr1a MO and HIGH Δfmr1 (LOW MO HIGH Δfmr1 Rescue) rescues the normal increase in cell number from 1 − 3 dfe (*p < 0.05, **p < 0.01). C, 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), and 1 μg/μl Δfmr1-t2A-eGFP + 0.1 mM fmr1a MO (HIGH MO HIGH Δfmr1 Rescue). Dashed lines outline the optic tectum. D, FMRP knockdown with HIGH fmr1a MO results in a negative percent change in cell number between 1 − 3 dfe, suggesting that proliferation and cell survival are affected with a higher concentration of morpholino. This decrease was rescued by co-electroporation of HIGH Δfmr1 (**p < 0.01, ***p < 0.001). E, A 2 h pulse of the thymidine analog CldU delivered at 3 dfe confirms that cell proliferation is decreased by HIGH fmr1a MO (*p < 0.05). F, Z-projections from in vivo confocal time-lapse images of Sox2bd::eGFP+ (Control) and 1 μg/μl Δfmr1-t2A-eGFP+ (HIGH FMRP OE) cells collected at 1 and 3 dfe. Dashed lines outline the optic tectum. G, H, The percent change in the number of eGFP+ cells increases over 3 d in control animals. HIGH FMRP OE significantly reduced the percent change in cell number between 1 − 3 dfe. Data from individual animals (G) and the mean ± SEM (H; ***p < 0.001). Scale bar, 50 μm.
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Figure 4: Knockdown and overexpression of FMRP decrease proliferation. A, Z-projections from in vivo confocal time-lapse images of cells expressing Sox2bd::eGFP + CMO (CMO) or 0.05 mM fmr1a MO (LOW fmr1a MO), and 1 μg/μl Δfmr1-t2A-eGFP + 0.05 mM fmr1a MO (LOW MO HIGH Δfmr1 Rescue) taken at 1 and 3 dfe. Dashed lines outline the optic tectum and inset shows a schematic of the optic tectum. B, The percent change in the number of eGFP+ cells increases over 3 d in CMO animals. FMRP knockdown with LOW fmr1a MO blocks the increase in cell number between 1 − 3 dfe. Coexpression of LOW fmr1a MO and HIGH Δfmr1 (LOW MO HIGH Δfmr1 Rescue) rescues the normal increase in cell number from 1 − 3 dfe (*p < 0.05, **p < 0.01). C, 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), and 1 μg/μl Δfmr1-t2A-eGFP + 0.1 mM fmr1a MO (HIGH MO HIGH Δfmr1 Rescue). Dashed lines outline the optic tectum. D, FMRP knockdown with HIGH fmr1a MO results in a negative percent change in cell number between 1 − 3 dfe, suggesting that proliferation and cell survival are affected with a higher concentration of morpholino. This decrease was rescued by co-electroporation of HIGH Δfmr1 (**p < 0.01, ***p < 0.001). E, A 2 h pulse of the thymidine analog CldU delivered at 3 dfe confirms that cell proliferation is decreased by HIGH fmr1a MO (*p < 0.05). F, Z-projections from in vivo confocal time-lapse images of Sox2bd::eGFP+ (Control) and 1 μg/μl Δfmr1-t2A-eGFP+ (HIGH FMRP OE) cells collected at 1 and 3 dfe. Dashed lines outline the optic tectum. G, H, The percent change in the number of eGFP+ cells increases over 3 d in control animals. HIGH FMRP OE significantly reduced the percent change in cell number between 1 − 3 dfe. Data from individual animals (G) and the mean ± SEM (H; ***p < 0.001). Scale bar, 50 μm.

Mentions: Evidence from humans suggests that the gene dosage of FMR1 is tightly regulated and that both decreases and increases in FMRP can cause disease. Individuals with decreases in FMRP with Fragile X Syndrome and individuals with gene duplications of FMR1 both present with intellectual disability (Rio et al., 2010; Nagamani et al., 2012; Vengoechea et al., 2012; Hickey et al., 2013). To test whether FMRP regulates cell proliferation and/or survival in the optic tectum, we manipulated FMRP expression levels using knockdown and overexpression. We electroporated animals with Sox2bd::eGFP and UAS::tRFPnls to label tectal progenitors and their progeny, and either CMO, LOW fmr1a MO, or HIGH fmr1a MO. Then, we performed in vivo time-lapse imaging of eGFP+ cells at 1, 2, and 3 dfe (Fig. 4A). We quantified the percent change in cell number between 1 and 3 dfe. CMO animals increased eGFP+ cell number from 1 − 3 dfe as NPCs proliferated. LOW fmr1a MO significantly reduced the normal increase in cell numbers seen in controls (Fig. 4B; CMO: N = 20 animals; LOW fmr1a MO: N = 17 animals, p = 0.0012g compared to CMO). This LOW fmr1a MO-mediated decrease in cell number was rescued by coexpression of 1 μg/μl Δfmr1-t2A-eGFP (Fig. 4B; LOW MO HIGH Δfmr1 Rescue: N = 10 animals, p = 0.035g compared to LOW fmr1a MO, p = 0.79g compared to CMO). When we knocked down FMRP using HIGH fmr1a MO, we found an even greater reduction in cell numbers, with a net loss of cells between 1-3 dfe (Fig. 4C,D; CMO: N = 24 animals; HIGH fmr1a MO: N = 24 animals, p < 0.0001h). This result suggests that FMRP knockdown with HIGH fmr1a MO increases cell death, consistent with our observation from the in vivo knockdown assay. The HIGH fmr1a MO-mediated decrease in cell number was rescued by coexpression of 1 μg/μl Δfmr1-t2A-eGFP (Fig. 4C,D; HIGH MO HIGH Δfmr1 Rescue: N = 17 animals, p = 0.0041h compared to HIGH fmr1 MO, p = 0.50h compared to CMO). These results demonstrate that fmr1a MO specifically knocks down FMRP since coexpression of MO-insensitive fmr1 was able to rescue the decrease in cell number.


FMRP regulates neurogenesis in vivo in Xenopus laevis tadpoles.

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

Knockdown and overexpression of FMRP decrease proliferation. A, Z-projections from in vivo confocal time-lapse images of cells expressing Sox2bd::eGFP + CMO (CMO) or 0.05 mM fmr1a MO (LOW fmr1a MO), and 1 μg/μl Δfmr1-t2A-eGFP + 0.05 mM fmr1a MO (LOW MO HIGH Δfmr1 Rescue) taken at 1 and 3 dfe. Dashed lines outline the optic tectum and inset shows a schematic of the optic tectum. B, The percent change in the number of eGFP+ cells increases over 3 d in CMO animals. FMRP knockdown with LOW fmr1a MO blocks the increase in cell number between 1 − 3 dfe. Coexpression of LOW fmr1a MO and HIGH Δfmr1 (LOW MO HIGH Δfmr1 Rescue) rescues the normal increase in cell number from 1 − 3 dfe (*p < 0.05, **p < 0.01). C, 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), and 1 μg/μl Δfmr1-t2A-eGFP + 0.1 mM fmr1a MO (HIGH MO HIGH Δfmr1 Rescue). Dashed lines outline the optic tectum. D, FMRP knockdown with HIGH fmr1a MO results in a negative percent change in cell number between 1 − 3 dfe, suggesting that proliferation and cell survival are affected with a higher concentration of morpholino. This decrease was rescued by co-electroporation of HIGH Δfmr1 (**p < 0.01, ***p < 0.001). E, A 2 h pulse of the thymidine analog CldU delivered at 3 dfe confirms that cell proliferation is decreased by HIGH fmr1a MO (*p < 0.05). F, Z-projections from in vivo confocal time-lapse images of Sox2bd::eGFP+ (Control) and 1 μg/μl Δfmr1-t2A-eGFP+ (HIGH FMRP OE) cells collected at 1 and 3 dfe. Dashed lines outline the optic tectum. G, H, The percent change in the number of eGFP+ cells increases over 3 d in control animals. HIGH FMRP OE significantly reduced the percent change in cell number between 1 − 3 dfe. Data from individual animals (G) and the mean ± SEM (H; ***p < 0.001). Scale bar, 50 μm.
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Figure 4: Knockdown and overexpression of FMRP decrease proliferation. A, Z-projections from in vivo confocal time-lapse images of cells expressing Sox2bd::eGFP + CMO (CMO) or 0.05 mM fmr1a MO (LOW fmr1a MO), and 1 μg/μl Δfmr1-t2A-eGFP + 0.05 mM fmr1a MO (LOW MO HIGH Δfmr1 Rescue) taken at 1 and 3 dfe. Dashed lines outline the optic tectum and inset shows a schematic of the optic tectum. B, The percent change in the number of eGFP+ cells increases over 3 d in CMO animals. FMRP knockdown with LOW fmr1a MO blocks the increase in cell number between 1 − 3 dfe. Coexpression of LOW fmr1a MO and HIGH Δfmr1 (LOW MO HIGH Δfmr1 Rescue) rescues the normal increase in cell number from 1 − 3 dfe (*p < 0.05, **p < 0.01). C, 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), and 1 μg/μl Δfmr1-t2A-eGFP + 0.1 mM fmr1a MO (HIGH MO HIGH Δfmr1 Rescue). Dashed lines outline the optic tectum. D, FMRP knockdown with HIGH fmr1a MO results in a negative percent change in cell number between 1 − 3 dfe, suggesting that proliferation and cell survival are affected with a higher concentration of morpholino. This decrease was rescued by co-electroporation of HIGH Δfmr1 (**p < 0.01, ***p < 0.001). E, A 2 h pulse of the thymidine analog CldU delivered at 3 dfe confirms that cell proliferation is decreased by HIGH fmr1a MO (*p < 0.05). F, Z-projections from in vivo confocal time-lapse images of Sox2bd::eGFP+ (Control) and 1 μg/μl Δfmr1-t2A-eGFP+ (HIGH FMRP OE) cells collected at 1 and 3 dfe. Dashed lines outline the optic tectum. G, H, The percent change in the number of eGFP+ cells increases over 3 d in control animals. HIGH FMRP OE significantly reduced the percent change in cell number between 1 − 3 dfe. Data from individual animals (G) and the mean ± SEM (H; ***p < 0.001). Scale bar, 50 μm.
Mentions: Evidence from humans suggests that the gene dosage of FMR1 is tightly regulated and that both decreases and increases in FMRP can cause disease. Individuals with decreases in FMRP with Fragile X Syndrome and individuals with gene duplications of FMR1 both present with intellectual disability (Rio et al., 2010; Nagamani et al., 2012; Vengoechea et al., 2012; Hickey et al., 2013). To test whether FMRP regulates cell proliferation and/or survival in the optic tectum, we manipulated FMRP expression levels using knockdown and overexpression. We electroporated animals with Sox2bd::eGFP and UAS::tRFPnls to label tectal progenitors and their progeny, and either CMO, LOW fmr1a MO, or HIGH fmr1a MO. Then, we performed in vivo time-lapse imaging of eGFP+ cells at 1, 2, and 3 dfe (Fig. 4A). We quantified the percent change in cell number between 1 and 3 dfe. CMO animals increased eGFP+ cell number from 1 − 3 dfe as NPCs proliferated. LOW fmr1a MO significantly reduced the normal increase in cell numbers seen in controls (Fig. 4B; CMO: N = 20 animals; LOW fmr1a MO: N = 17 animals, p = 0.0012g compared to CMO). This LOW fmr1a MO-mediated decrease in cell number was rescued by coexpression of 1 μg/μl Δfmr1-t2A-eGFP (Fig. 4B; LOW MO HIGH Δfmr1 Rescue: N = 10 animals, p = 0.035g compared to LOW fmr1a MO, p = 0.79g compared to CMO). When we knocked down FMRP using HIGH fmr1a MO, we found an even greater reduction in cell numbers, with a net loss of cells between 1-3 dfe (Fig. 4C,D; CMO: N = 24 animals; HIGH fmr1a MO: N = 24 animals, p < 0.0001h). This result suggests that FMRP knockdown with HIGH fmr1a MO increases cell death, consistent with our observation from the in vivo knockdown assay. The HIGH fmr1a MO-mediated decrease in cell number was rescued by coexpression of 1 μg/μl Δfmr1-t2A-eGFP (Fig. 4C,D; HIGH MO HIGH Δfmr1 Rescue: N = 17 animals, p = 0.0041h compared to HIGH fmr1 MO, p = 0.50h compared to CMO). These results demonstrate that fmr1a MO specifically knocks down FMRP since coexpression of MO-insensitive fmr1 was able to rescue the decrease in cell number.

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