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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 dendritic development. In vivo two-photon time-lapse images of cells expressing Sox2bd::eGFP and CMO or fmr1a MO collected at 2 and 3 dfe. A, Two-photon Z-projections of imaged cells and their reconstructed dendritic arbors at 2 and 3 dfe for cells with FMRP knockdown compared to control. B, HIGH (0.1 mM) fmr1a MO decreased total dendritic length at 3 dfe (**p < 0.01). C, HIGH fmr1a MO decreased total dendritic branch tip number at 2 and 3 dfe (*p < 0.05, **p < 0.01). D, Branch density was unchanged between the groups. E, Two-photon Z-projection and reconstructed dendritic arbor of a cell expressing 1 μg/μl Δfmr1-t2A-eGFP (HIGH FMRP OE) at 3 dfe. F, G, HIGH FMRP OE decreased total dendritic length (F) and total dendritic branch tip number (G) compared to control (Sox2bd::eGFP; ***p < 0.001). H, Two-photon Z-projections of imaged cells and their reconstructed dendritic arbors at 3 dfe for cells when FMRP is knocked down (HIGH fmr1a MO), overexpressed with 0.5 μg/μl Δfmr1-t2A-eGFP (LOW FMRP OE), and rescued (HIGH MO LOW Δfmr1 Rescue) compared to control (CMO). I, J, Co-electroporation of LOW Δfmr1-t2A-eGFP rescued HIGH fmr1a MO-mediated decreases in total dendritic length (I) and dendritic branch tip number (J) (*p < 0.05, **p <0.01). Scale bars, 20 μm.
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Figure 7: FMRP regulates dendritic development. In vivo two-photon time-lapse images of cells expressing Sox2bd::eGFP and CMO or fmr1a MO collected at 2 and 3 dfe. A, Two-photon Z-projections of imaged cells and their reconstructed dendritic arbors at 2 and 3 dfe for cells with FMRP knockdown compared to control. B, HIGH (0.1 mM) fmr1a MO decreased total dendritic length at 3 dfe (**p < 0.01). C, HIGH fmr1a MO decreased total dendritic branch tip number at 2 and 3 dfe (*p < 0.05, **p < 0.01). D, Branch density was unchanged between the groups. E, Two-photon Z-projection and reconstructed dendritic arbor of a cell expressing 1 μg/μl Δfmr1-t2A-eGFP (HIGH FMRP OE) at 3 dfe. F, G, HIGH FMRP OE decreased total dendritic length (F) and total dendritic branch tip number (G) compared to control (Sox2bd::eGFP; ***p < 0.001). H, Two-photon Z-projections of imaged cells and their reconstructed dendritic arbors at 3 dfe for cells when FMRP is knocked down (HIGH fmr1a MO), overexpressed with 0.5 μg/μl Δfmr1-t2A-eGFP (LOW FMRP OE), and rescued (HIGH MO LOW Δfmr1 Rescue) compared to control (CMO). I, J, Co-electroporation of LOW Δfmr1-t2A-eGFP rescued HIGH fmr1a MO-mediated decreases in total dendritic length (I) and dendritic branch tip number (J) (*p < 0.05, **p <0.01). Scale bars, 20 μm.

Mentions: The in vivo imaging experiments above suggested that neuronal dendrite arbor development might be abnormal with knockdown or overexpression of FMRP. Defects in spine morphology have been widely reported in Fragile X patients and animal models (Hinton et al., 1991; Comery et al., 1997; Irwin et al., 2001; Nimchinsky et al., 2001; Cruz-Martín et al., 2010), but reports of defects in dendritic morphology have been mixed (Irwin et al., 2002; Galvez et al., 2003; Lee et al., 2003; Castrén et al., 2005; Koekkoek et al., 2005; Thomas et al., 2008; Guo et al., 2011; Scotto-Lomassese et al., 2011; Sheridan et al., 2011; Guo et al., 2012; Till et al., 2012; Telias et al., 2013; Doers et al., 2014). While Xenopus tectal neurons lack dendritic spines, we analyzed dendritic arbor morphology to assess whether FMRP plays a role in dendritic development. We imaged tectal neurons in vivo in animals sparsely electroporated with Sox2bd::eGFP and either CMO, LOW fmr1a MO, or HIGH fmr1a MO at 2 and 3 dfe using a two-photon microscope (Fig. 7A). We reconstructed the dendritic arbors of imaged neurons and quantified total dendritic branch length and total dendritic branch tip number (Fig. 7A−C). At 2 dfe, HIGH fmr1a MO decreased total dendritic branch tip number (Fig. 7C; 2 dfe Branch tip number: CMO N = 66 cells; HIGH fmr1a MO N = 46 cells, p = 0.018oo). At 2 dfe there were also noticeable decreases in total dendritic branch length with both MO concentrations and in total dendritic branch tip number with LOW fmr1a MO, but these did not reach significance (Fig. 7B,C; 2 dfe Length: CMO N = 66 cells; LOW fmr1a MO N = 60 cells, p = 0.34pp compared to CMO; HIGH fmr1a MO N = 46 cells, p = 0.20pp compared to CMO; 2 dfe Branch tip number: LOW fmr1a MO N = 60 cells, p = 0.12oo compared to CMO). At 3 dfe, HIGH fmr1a MO decreased total dendritic branch length and total dendritic branch tip number (Fig. 7B,C; 3 dfe Length: CMO N = 68 cells; HIGH fmr1a MO N = 49 cells, p = 0.0097qq; 3 dfe Branch tip number: CMO N = 68 cells; HIGH fmr1a MO N = 49 cells, p = 0.0014rr). We calculated branch density as the ratio of total dendritic branch tip number/total dendritic branch length and found no change in branch density with FMRP knockdown (Fig. 7D). This suggests that neurons lacking FMRP follow the same branching rule as control cells, they are just smaller overall.


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 dendritic development. In vivo two-photon time-lapse images of cells expressing Sox2bd::eGFP and CMO or fmr1a MO collected at 2 and 3 dfe. A, Two-photon Z-projections of imaged cells and their reconstructed dendritic arbors at 2 and 3 dfe for cells with FMRP knockdown compared to control. B, HIGH (0.1 mM) fmr1a MO decreased total dendritic length at 3 dfe (**p < 0.01). C, HIGH fmr1a MO decreased total dendritic branch tip number at 2 and 3 dfe (*p < 0.05, **p < 0.01). D, Branch density was unchanged between the groups. E, Two-photon Z-projection and reconstructed dendritic arbor of a cell expressing 1 μg/μl Δfmr1-t2A-eGFP (HIGH FMRP OE) at 3 dfe. F, G, HIGH FMRP OE decreased total dendritic length (F) and total dendritic branch tip number (G) compared to control (Sox2bd::eGFP; ***p < 0.001). H, Two-photon Z-projections of imaged cells and their reconstructed dendritic arbors at 3 dfe for cells when FMRP is knocked down (HIGH fmr1a MO), overexpressed with 0.5 μg/μl Δfmr1-t2A-eGFP (LOW FMRP OE), and rescued (HIGH MO LOW Δfmr1 Rescue) compared to control (CMO). I, J, Co-electroporation of LOW Δfmr1-t2A-eGFP rescued HIGH fmr1a MO-mediated decreases in total dendritic length (I) and dendritic branch tip number (J) (*p < 0.05, **p <0.01). Scale bars, 20 μm.
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Figure 7: FMRP regulates dendritic development. In vivo two-photon time-lapse images of cells expressing Sox2bd::eGFP and CMO or fmr1a MO collected at 2 and 3 dfe. A, Two-photon Z-projections of imaged cells and their reconstructed dendritic arbors at 2 and 3 dfe for cells with FMRP knockdown compared to control. B, HIGH (0.1 mM) fmr1a MO decreased total dendritic length at 3 dfe (**p < 0.01). C, HIGH fmr1a MO decreased total dendritic branch tip number at 2 and 3 dfe (*p < 0.05, **p < 0.01). D, Branch density was unchanged between the groups. E, Two-photon Z-projection and reconstructed dendritic arbor of a cell expressing 1 μg/μl Δfmr1-t2A-eGFP (HIGH FMRP OE) at 3 dfe. F, G, HIGH FMRP OE decreased total dendritic length (F) and total dendritic branch tip number (G) compared to control (Sox2bd::eGFP; ***p < 0.001). H, Two-photon Z-projections of imaged cells and their reconstructed dendritic arbors at 3 dfe for cells when FMRP is knocked down (HIGH fmr1a MO), overexpressed with 0.5 μg/μl Δfmr1-t2A-eGFP (LOW FMRP OE), and rescued (HIGH MO LOW Δfmr1 Rescue) compared to control (CMO). I, J, Co-electroporation of LOW Δfmr1-t2A-eGFP rescued HIGH fmr1a MO-mediated decreases in total dendritic length (I) and dendritic branch tip number (J) (*p < 0.05, **p <0.01). Scale bars, 20 μm.
Mentions: The in vivo imaging experiments above suggested that neuronal dendrite arbor development might be abnormal with knockdown or overexpression of FMRP. Defects in spine morphology have been widely reported in Fragile X patients and animal models (Hinton et al., 1991; Comery et al., 1997; Irwin et al., 2001; Nimchinsky et al., 2001; Cruz-Martín et al., 2010), but reports of defects in dendritic morphology have been mixed (Irwin et al., 2002; Galvez et al., 2003; Lee et al., 2003; Castrén et al., 2005; Koekkoek et al., 2005; Thomas et al., 2008; Guo et al., 2011; Scotto-Lomassese et al., 2011; Sheridan et al., 2011; Guo et al., 2012; Till et al., 2012; Telias et al., 2013; Doers et al., 2014). While Xenopus tectal neurons lack dendritic spines, we analyzed dendritic arbor morphology to assess whether FMRP plays a role in dendritic development. We imaged tectal neurons in vivo in animals sparsely electroporated with Sox2bd::eGFP and either CMO, LOW fmr1a MO, or HIGH fmr1a MO at 2 and 3 dfe using a two-photon microscope (Fig. 7A). We reconstructed the dendritic arbors of imaged neurons and quantified total dendritic branch length and total dendritic branch tip number (Fig. 7A−C). At 2 dfe, HIGH fmr1a MO decreased total dendritic branch tip number (Fig. 7C; 2 dfe Branch tip number: CMO N = 66 cells; HIGH fmr1a MO N = 46 cells, p = 0.018oo). At 2 dfe there were also noticeable decreases in total dendritic branch length with both MO concentrations and in total dendritic branch tip number with LOW fmr1a MO, but these did not reach significance (Fig. 7B,C; 2 dfe Length: CMO N = 66 cells; LOW fmr1a MO N = 60 cells, p = 0.34pp compared to CMO; HIGH fmr1a MO N = 46 cells, p = 0.20pp compared to CMO; 2 dfe Branch tip number: LOW fmr1a MO N = 60 cells, p = 0.12oo compared to CMO). At 3 dfe, HIGH fmr1a MO decreased total dendritic branch length and total dendritic branch tip number (Fig. 7B,C; 3 dfe Length: CMO N = 68 cells; HIGH fmr1a MO N = 49 cells, p = 0.0097qq; 3 dfe Branch tip number: CMO N = 68 cells; HIGH fmr1a MO N = 49 cells, p = 0.0014rr). We calculated branch density as the ratio of total dendritic branch tip number/total dendritic branch length and found no change in branch density with FMRP knockdown (Fig. 7D). This suggests that neurons lacking FMRP follow the same branching rule as control cells, they are just smaller overall.

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